Method and composition for altering a b cell mediated pathology

ABSTRACT

The present invention provides compositions for altering a B cell mediated pathology in a patient. The compositions may comprise at least one and/or two chimeric proteins. Each chimeric protein comprises at least a portion of either the VH or VL region of an immunoglobulin module from particular B cells from a patient having a B cell mediated pathology, and an immunoglobulin constant region. The genes encoding VH and/or VL regions and the genes encoding immunoglobulin constant regions are isolated and inserted in an expression vector. The chimeric proteins may be produced by introducing the expression vectors into insect cell lines. Proteins may be further purified and conjugated to a compound such as a immunogenic carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 13/293,495 filedNov. 10, 2011, which is a Continuation of U.S. Ser. No. 10/843,703 filedMay 10, 2004 now U.S. Pat. No. 8,114,404 issued Feb. 14, 2012, which isa Continuation of U.S. Ser. No. 09/927,121 filed on Aug. 10, 2001, nowU.S. Pat. No. 6,911,204 issued Jun. 28, 2005, which claims priority tothe U.S. Provisional Application No. 60/224,723 filed Aug. 11, 2000,U.S. Provisional Application No. 60/224,722 filed on Aug. 11, 2000 andU.S. Provisional Application No. 60/279,079 filed on Mar. 23, 2001, allof which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of immunology andimmunotherapy. More specifically, this invention relates to methods andcompositions for altering B cell mediated pathologies, such as B cellmalignancies and/or autoimmune diseases.

BACKGROUND OF THE INVENTION

The immune system produces both antibody-mediated and cell-mediatedresponses. Each type of immune response is regulated by a type oflymphocyte, B cells (for antibody-mediated response) and T cells (forcell-mediated response). B cells initially recognize an antigen when theantigen binds to the IgM and IgD molecules on the B cell's surface. EachB cell clone recognizes only specific antigens due to the uniqueidiotype of that clone. Upon recognition of the antigen, B cellsinternalize and process the antigen for presentation via MHC class IImolecules. B cells can thereby function as an antigen presenting cell(“APC”) for T cells. T cells bind to portions of foreign proteins(antigens) when portions of the protein associate with a majorhistocompatibility complex molecule (“MHC”), typically on an APC, inwhich the antigen is digested into fragments and presented on thesurface of the APC bound to its MHC.

Several types of cancers have their origin in the circulatory system.Among the major types are: leukemias, a neoplasm of the bone marrow andblood; myelomas, a cancer of B cells; and lymphomas, a group of cancersthat originate in the lymphatic system. Lymphomas can be furtherclassified into several groups; one of these groups is the non-Hodgkin'slymphomas which, in turn, forms a diverse group of cancers. Three broadcategories of these lymphomas are defined according to the InternationalWorking Formulation for tumor classification, low grade, intermediategrade and high grade, which differ in their curability andaggressiveness (Cheson, et al., “Report of an International Workshop toStandardize Response Criteria for Non-Hodgkin's Lymphomas,” J. ClinOncol. 17(4):1244, 1999). Overall, these lymphomas collectively rankfifth in the United States in terms of cancer incidence and mortality,and approximately 50,000 new cases are diagnosed each year.

In a recent study which examined fifty-one case isolates of high-gradenon-Hodgkins's lymphoma (NHL), forty-three were shown to be derived fromB cells while eight were shown to be derived from T cells (Brown et al.,Histopathology 14:621-27, 1989). Therefore, treatments directedspecifically towards pathological B cells would be valuable in thetreatment of non-Hodgkin's lymphomas and myelomas.

Initial attempts in the field to develop an immunology-based treatmentdirected at antigens uniquely produced by malignant B cells involvedlaboriously isolating and purifying idiotypic (Id) proteins directlyfrom the pathological B cells. This purified protein was first used inmodel systems to treat the associated lymphoma. It was demonstrated thatthis active immunization against idiotypic determinants on isolatedproteins could produce resistance to tumor growth in a mouse modelsystem (Daley et al., J. Immunol. 120(5):1620-24, 1978; Sakato et al.,Microbiol Immunol. 23(9):927-31, 1979). This phenomenon of resistance totumor growth has been subsequently reproduced in a number of additionalexperimental tumor models (Stevenson et al., J. Immunol. 130(2):970-03,1983; George et al., J. Immunol. 141(6):2168-74, 1988; Kwak, et al.,Blood 76(11):2411-17, 1990).

Among the first attempts at bringing this idea and technology into theclinic was very labor intensive and utilized mouse monoclonal antibodiesgenerated against proteins isolated from the patients' individuallymphomas following biopsy. Meeker and coworkers generated mousemonoclonal anti-idiotype antibodies for treatment of eleven patientsafter most had already undergone conventional lymphoma therapy (Meekeret al., Blood 65:1349-63, 1985). Positive results were obtained inroughly half the patients, with one case of apparent remission. In someof the patients, however, the lymphoma cells developed a resistance tothe antibody via switching the class of cell surface-expressedantibodies (Meeker et al., N. Engl J. Med. 312:1658-65, 1985).

Another way a B cell lymphoma clone developed resistance toanti-idiotypic antibodies is via a somatic mutation in the CDR2 region(Cleary et al., Cell 44:97-106, 1986), thereby evading recognition.While this passive immunity approach for treatment has the advantagethat it only requires isolation and purification of a relatively minoramount of idiotypic protein from a patient for raising an immuneresponse in a mouse, the usefulness for treating lymphomas withmonoclonal antibodies directed at idiotypes is limited. In the absenceof a robust and convenient way to produce large quantities of idiotypicprotein, however, this could prove to be the only practical way toexploit the abilities of the immune system to directly attack theidiotype of a B cell lymphoma.

Kwak et al. pursued a different approach and attempted the activeimmunization of patients using proteins purified from their own uniquelymphomas in spite of the logistical requirement for isolating largequantities of idiotypic proteins (Kwak et al., N. Engl, J. Med.327:1209-15, 1992). Patients who had minimal or no disease followingchemotherapy were treated by vaccination with autologous idiotypeproteins. In order to obtain sufficient quantities of idiotypic proteinsfor vaccination, lymphoma cells obtained by biopsy were fused with anestablished cell line to facilitate their growth in tissue culture, andthe secreted idiotype proteins were purified via chromatography. Largescale application of this method of immunization is precluded due to theextreme labor requirements, technical barriers, and prohibitive costs.Additionally, concerns have recently been raised concerning the viralloads associated with protein production in mammalian cells.

In a following paper, Hsu et al. reported on the phase I/II of the aboveclinical trial utilizing vaccination of the idiotype conjugated tokeyhole limpet hemocyanin (KLH) in the treatment of B-cell lymphoma (Hsuet al., Blood 89:3129-35, 1997). After standard chemotherapy, 41patients with refractory non-Hodgkin's B-cell lymphoma were vaccinatedwith a tumor-specific idiotype. As per Kwak et al (1992), supra, thetumor-specific idiotype antigens were obtained by chromatographicpurification of proteins produced by the patients' hybridomas. Theseproteins were therefore composed of the entire variable and constantregions of the patient's own immunoglobulin from the patients'lymphomas. The results showed that the generation of an anti-idiotyperesponse correlated with improved clinical outcome. The duration offreedom from disease progression and overall survival of all patientsmounting an anti-idiotype cellular immune response were significantlyprolonged compared to those patients who did not mount an immuneresponse. This study confirms that patients with B-cell lymphomas can beinduced to make a specific immune response against tumor idiotype (Id)protein. Furthermore, the ability to generate an anti-idiotype immuneresponse correlates with a more favorable clinical outcome. However, totreat each individual patient, lymphoma cells obtained by biopsy must befused to established cell lines in order to allow the production ofsufficient protein to vaccinate a typical patient. This process would-bedifficult or impractical to use on a commercial scale.

More recently, Bendandi et al. demonstrated idiotypic, patient-specificvaccination-induced remissions in patients with follicular lymphoma(Bendandi et al., Nat. Med. 5:1171-77, 1999). Following standardchemotherapy, twenty patients demonstrating complete clinical remissionwere vaccinated using patient-specific idiotypic proteins accompanied bygranulocyte-monocyte colony-stimulating factor (GM-CSF; see infra.).Molecular analysis of the translocations characteristic of this lymphomawas conducted prior to chemotherapy, at clinical remission andfollowing, vaccination therapy. Eight of eleven patients withdetectable, translocations after chemotherapy-induced remission werefound to undergo complete molecular remission following thisvaccination. Tumor-specific cytotoxic CD8+ and CD4+ T cells were foundin 19 of 20 patients. Tumor-specific antibodies were also detected butwere not found to be required for remission. Again, this study usedidiotypic proteins made up of the entire variable and constant region ofthe immunoglobulin found associated with the patient's lymphoma andproduced by heterohybridoma fusion.

Therefore, directing an immune response to the idiotype of cells is apromising approach, but the above techniques are limited by therequirement of producing sufficient quantities of idiotypic proteinsfrom each patient's lymphoma cells.

The concept of anti-idiotypic immunity against B cell tumors has alsobeen used in the case of multiple myeloma. Results have been reported byKwak and coworkers regarding its use in enhancing the specific efficacyof allogeneic marrow grafts by pre-immunizing the donor with myeloma IgGisolated from the patient (Kwak et al., Lancet 345 (8956):1016-20,1995). Also, Massaia and coworkers vaccinated patients in remissionfollowing high-dose chemotherapy, followed by peripheral blood stem celltransplantation (Massaia et al., Blood 94:673-83, 1999).

Granulocyte-monocyte colony-stimulating factor (GM-CSF), used above inBendandi et al.'s study, is a hematopoietic growth factor whichstimulates proliferation and differentiation of hematopoietic progenitorcells. This cytokine also plays a role in shaping cellular immunity byaugmenting T-cell proliferation (Santoli et al., J. Immunol.141(2):519-26, 1988). increasing expression of adhesion molecules ongranulocytes and monocytes (Young et al., J. Immunol 145(2):607-15,1990; Grabstein et al., Science 232(4749):506-08, 1986), and augmentingantigen presentation (Morrissey et al., J. Immunol. 139(4):1113-9, 1987;Heufler et al., J. Exp. Med. 167(2):700-05, 1988; Smith et al., J.Immunol. 144(5):1777-82, 1990).

Cell-based vaccines genetically engineered to produce GM-CSF have beenshown to induce cellular immune responses capable of eliminatingsystemic lymphomas in preclinical models. This effect is mediatedexclusively through activation of the cellular arm of the immune system(Levitsky et al., J. Immuno. 156(10): 3858-65, 1996). Similarly, lowdoses of free GM-CSF have been shown to enhance the protectiveanti-tumor immunity induced by idiotype protein-KLH immunization becauseof its ability to enhance immunity through an effect on the CD8 cells(Kwak et al., Proc. Natl. Acad. Sci. USA 93(20):10972-77, 1996. In onestudy, GM-CSF was shown to be the best immunomodulator to generateanti-tumor immunity among those tested in a model system (Dranoff, G.,Proc. Natl. Acad. Sci. USA 90(8):3539-43, 1993.)

GM-CSF has also been used as a portion of a chimeric protein used togenerate an immune response in model systems. Chen and Levy (Chen andLevy, J. Immunol. 154(7):3105-17, 1995; U.S. Pat. No. 6,099,846) studiedthe production of mouse monoclonal antibodies using a chimeric proteincontaining a portion of GM-CSF plus a portion of an antigen of interest,namely an idiotypic region obtained from a murine B-cell tumor, 38C13,both fused to portions of human immunoglobulin chains. Chen andcoworkers have also studied fusion proteins where the GM-CSF moiety hasbeen replaced by portions of IL-2 or IL-4 (Chen et al., J. Immunol.153(10):4775-87, 1994). One explanation for the requirement of includingthe GM-CSF moiety (or interleukin moiety) was to augment the effect-oflow levels of chimeric protein produced by the mammalian cell expressionsystem. However, the use of purified GM-CSF co-administered with achimeric protein to enhance the immune response of a vaccination has notbeen demonstrated.

With the advent of recombinant DNA technology, heavy and light chaincDNA molecules can now be cloned from hybridomas or from combinatoriallibraries employing the polymerase chain reaction (PCR). Thisrecombinant DNA technology allows researchers to manipulate the effectorfunction or the binding function of a selected monoclonal antibody. Inaddition, combinatorial libraries of immunoglobulins can be generated bycloning a large number of V_(L) and V_(H) genes, randomly assorting themto create a library of different binding specificities, expressing themin E. coli, then screening the stochastic library for clones with thedesired binding affinities (Huse et al., Science 246(4935):1275-81,1989). Using this recombinant approach, human antibodies were clonedwith high affinity and specificity for tetanus toxoid from a randomizedcombinatorial library expressed in E. coli (Mullinax et al., Proc. Natl.Acad. Sci. 87(20):8095-99, 1990). The immunoglobulin genes were clonedfrom activated B-cells into bacteriophage vectors using the polymerasechain reaction (PCR) with specific primers. The H and L chains wererandomly combined and co-expressed in E. coli to comprise a library of 1members. This combinatorial library was screened with ¹²⁵I-tetanustoxoid and 0.2% of the clones displayed binding activity (Mullinax etal., supra). In addition, murine monoclonal antibodies have also beenidentified using a similar approach (Huse et al., supra; Caton et al.,Proc. Natl. Acad. Sci. 87(16):6450-54, 1990). Winter and co-workers useda plasmid vector to clone immunoglobulin domains by the polymerase chainreaction for expression in bacteria (Orlandi et al., Proc. Natl. Acad.Sci. 86(10):3833-37, 1989).

Newly developed E. coli antibody cloning systems are very useful for theidentification of genes encoding desired binding specificities. However,antibodies produced in E. coli are not generally useful for therapeuticapplications. Typically, only the antibody antigen binding fragments,Fab or Fv, can be produced as secreted products in bacteria. In the rareinstance when a whole chain tetrameric IgG has been produced in E. coli,the CH2 domains are not glycosylated. Nonglycosylated antibodies lackthe cytolytic activities antibody-directed cellular cytotoxicity (ADCC)and complement activation that make passive immunotherapy so powerful.Mammalian expression systems produce glycosolated antibody and thuscircumvent this limitation of the bacterial system. However, recentmodifications in the CBER division of the FDA's “Points to Consider”clearly signal their concerns about viral loads associated withmonoclonal antibodies produced in mammalian cells. Moreover, it isexpected that any engineered antibody produced in a mammalian expressionsystem will be quite expensive ($1500-$5000 per dose). Alternativeexpression systems that circumvent the difficulties encountered withcurrent mammalian and bacterial systems are therefore highly desirable.

The baculovirus expression system is an attractive alternative toantibody production in E. coli and mammalian cells. The expression ofrecombinant proteins using the baculovirus system has been demonstratedin the past several years and has emerged as an excellent choice forhigh yield production (1-100 mg/L) of biologically active proteins ineukaryotic cells. The baculovirus/insect cell system also circumventsthe solubility problems often encountered when recombinant proteins areoverexpressed in prokaryotes. In addition, insect cells contain theeukaryotic post-translational modification machinery responsible forcorrect folding, disulfide formation, glycosylation, β-hydroxylation,fatty acid acylation, prenylation, phosphorylation and amidation notpresent in prokaryotes. The production of a functional, glycosylatedmonoclonal antibody recognizing human colorectal carcinoma cells from abaculovirus expression system has been recently demonstrated (Nesbit, J.Immunol. Methods 151:201-208, 1992). Additionally, expression ofrecombinant IgA has also been demonstrated in baculovirus cells, andthis IgA was correctly assembled into heavy chain/light chainheterodimers, N-glycosylated, and secreted (Carayannopoulos et al.,Proc. Natl. Acad. Sci. 91:8348-52, 1994, PCT Publication No. WO98/30577, U.S. Pat. No. 6,063,905). However, the use of baculovirus toexpress a chimeric idiotypic protein for use as an immunotherapeuticagent to modify a B cell pathology such as B cell malignancies andautoimmune diseases has not been demonstrated.

SUMMARY OF THE INVENTION

The present invention provides a method for altering a B cell mediatedpathology in a patient. This method includes administering a compositionthat contains at least one chimeric protein having at least a portion ofa V_(L) and V_(H) region of an immunoglobulin variable region and atleast a portion of an immunoglobulin constant region. The V_(L) andV_(H) region used in this composition is associated with a particularimmunoglobulin produced by a B cell from a patient having a B cellmediated pathology. After administering such a composition into apatient, the B cell mediated pathology in the patient is altered.

The present invention also provides a method for altering a B cellmediated pathology in a patient by administering a compositioncontaining two different chimeric proteins. Each chimeric protein has atleast a portion of a V_(L) and/or V_(H) region of an immunoglobulinchain linked to at least a portion of an immunoglobulin constant region.The V_(L) and/or V_(H) regions that are part of the chimeric protein areassociated with particular immunoglobulin chains from a B cell of thepatient having a B cell mediated pathology.

Specific immunoglobulin chains containing patient-derived unique V_(L)and/or V_(H) chains can be developed as therapeutic compositions. Theywill have therapeutic value for patients suffering from a variety of Bcell malignancies or autoimmune diseases. Among the major types ofcancers that can be treated are leukemias, myelomas, and lymphomas;among the lymphomas is non-Hodgkin's lymphoma. As an example of thetherapeutic value of the instant invention, antigens derived from B celllymphomas have been used to treat patients.

Suspected self-antigens can be used to affinity purify B cells involvedin autoimmune diseases, such as multiple sclerosis (MS) (Warren andCatz, Mult. Scler. 6(5):300-11, 2000), systemic lupus erythematosus(SLE) (Zhang, J. et al., J. Immunol. 166(1):6-10, 2001; Odendahl, M. etal., J. Immunol. 165(10):5970-79, 2000), anti-Hu associatedparaneoplastic neurological syndromes (Rauer, S. and Kaiser, R., J.Neuroimmunol. 111(1-2):241-44, 2000); and autoimmune hepatitis (AIH)(Ogawa, S. et al., J. Gastroenterol. Hepatol (1):69-75, 2000). Otherautoimmune diseases which may have B cell involvement include rheumatoidarthritis (RA), myasthenia gravis (MG), autoimmune thyroiditis(Hashimoto's thyroiditis), autoimmune uveoretinitis, polymyositis,scleroderma, and certain types of diabetes. Following the purificationof a small number of pathogenic B cells, the variable portion of theimmunoglobulins expressed by these cells may be cloned via PCR using themethods described in the invention. Once cloned, the V_(L) and/or V_(H)portions of the immunoglobulin chains specifically involved in the Bcell pathology can be used to make chimeric proteins which can beexpressed in a baculovirus system as described herein.

The immunoglobulin constant regions used in the above compositions andchimeric protein can be from IgG₁, IgG₂, IgG₃, IgG₄ IgA₁, IgA₂, IgM,IgD, IgE heavy chains, and K or λ light chains or portions thereof. Insome of the embodiments, the chimeric protein only contains either theV_(L) and/or V_(H) region of an immunoglobulin region with animmunoglobulin constant region. Examples of chimeric proteins includeV_(H)-IgG₆₅ 1, V_(L)-K, and V_(L)-λ. In another embodiment, thecomposition contains two chimeric proteins that each respectivelycontains a V_(L) and V_(H) region with an immunoglobulin constantregion. Examples include V_(H)-IgG_(γ1) and V_(L)-K, and V_(H)-IgG_(γ1)and V_(L)-λ.

The present invention also provides a method for producing chimericproteins using recombinant DNA technology and an expression system. Thismethod includes the following steps: (a) isolating genes encoding theV_(L) or V_(H) region of an immunoglobulin chain from B cells of apatient having a B cell mediated pathology, (b) inserting the isolatedgene encoding the V_(L) or V_(H) region of an immunoglobulin chain andthe gene encoding an immunoglobulin constant region into an expressionvector to allow the expression of a chimeric protein, (c) producing thechimeric protein by introducing the expression vector into insect celllines and allowing its expression, and (d) isolating the chimericprotein. The method for producing chimeric proteins further includes astep of inserting a gene encoding either the V_(L) and/or V_(H) regionof an immunoglobulin chain and a gene encoding a second immunoglobulinconstant region into the expression vector to allow the expression ofthe second chimeric protein.

The present invention further provides a composition for altering a Bcell mediated pathology in a patient. This composition contains at leastone chimeric protein having at least a portion of a V_(L) and/or V_(H)region of an immunoglobulin chain and at least a portion of animmunoglobulin constant region. In preferred embodiments, the chimericproteins may comprise at least a portion of a V_(H) region of animmunoglobulin chain and at least a portion of an immunoglobulinconstant region. The V_(L) or V_(H) region that is part of the chimericprotein are associated with a particular immunoglobulin chain from a Bcell of a patient having a B cell mediated pathology. The compositionfurther contains a second chimeric protein having at least a portion ofa V_(L) and/or V_(H) region of an immunoglobulin chain and at least aportion of a second immunoglobulin constant region. In other preferredembodiments, the second chimeric protein may comprise at least a portionof a V_(L) or V_(H) region of an immunoglobulin chain and at least aportion of an immunoglobulin constant region. The V_(L) or V_(H) regionthat is part of the chimeric protein is associated with a particularimmunoglobulin chain from a B cell of a patient having a B cell mediatedpathology.

In one of the embodiments of the invention, the composition comprisestwo chimeric proteins. The first of the chimeric protein comprises theentire V_(H) region and a human constant region of an immunoglobulinIgG_(γ1) (V_(H)-IgG_(γ1)), and the second chimeric protein comprises theentire V_(L) and a human K or λ constant region (V_(L)-C_(K) orV_(L)-C_(λ)). In other preferred embodiments, either or both of thechimeric proteins may comprise at least a portion of a V_(L) and/orV_(H) region of an immunoglobulin chain, plus a linker region, and atleast a portion of an immunoglobulin constant region.

In another embodiment of the invention, the composition contains asingle chimeric protein containing either a V_(L) and/or V_(H) regionfrom a particular immunoglobulin chain from a B cell of a patient and animmunoglobulin constant region. Examples include chimeric proteinsV_(H)-IgG_(γ1), V_(L)-K, V_(L)-λ, V_(L)-IgG_(γ1), V_(H)-K, and V_(H)-λ.

In one of the embodiments of the invention, the expression vector usedto express the chimeric proteins is a baculovirus vector. The vectorpreferably contains two expression cassettes each having a promoter, asecretory signal sequence and a chimeric protein. One expressioncassette contains the baculovirus AcNPV p10 promotor linked to the honeybee melittin secretory signal sequence. The other expression cassettehas the polyhedrin promotor linked to a human placental alkalinephosphatase secretory-signal sequence. In addition to the listedpromoters and signal sequences, other promoters and signal sequencesknown to those skilled in the art could be used. In some preferredembodiments, the signal sequences are endogenous signal sequencesassociated with the V_(L) and V_(H) genes isolated from patients, orother signal sequences involved in antibody production. The genesencoding the V_(L) or V_(H) portions of the immunoglobulin chains, andthe genes encoding immunoglobulin constant region are inserted,separately and/or together, into the above expression cassette of thebaculovirus vector allowing expression of one or two chimeric proteins.In a preferred embodiment, the constant region of the immunoglobulinheavy chain, such as IgG_(γ1), with either the V_(L) or V_(H) region, iscontrolled by the polyhedrin promotor.

Chimeric proteins produced are purified using affinity columns withanti-immunoglobulin antibodies or Ig-binding proteins, such as protein Afor the constant, region of an immunoglobulin heavy chain, and protein Lfor kappa light chains, and/or any other proteins that bind to animmunoglobulin binding domain.

The present invention also contemplates covalently coupling the chimericproteins to a carrier protein such as keyhole limpet hemocyanin (KLH).The composition of the present invention may also be administered into apatient together with a cytokine such as granulocyte-macrophage-CS-F(GM-CSF), or a chemokine such as a monocyte chemotactic protein 3 (MCP3). Because the present composition of the present invention containingchimeric protein(s) is specifically related to a particularimmunoglobulin from B cells of a patient having B cell mediatedpathology, administration of this composition induces an immune responseagainst the disease specific idiotype in which particular V_(L) or V_(H)segments are involved. Similarly, responses against B cells associatedwith autoimmune diseases involving B cells that use a restrictedrepertoire of immunoglobulin V-region segments, such as V_(L) or V_(H)segments may induce a therapeutic result. Thus, the administration ofthe composition of the present invention alters a B cell mediatedpathology and/or autoimmune diseases in a patient. The administrationroutes for the invented composition include but are not limited to oraldelivery, inhalation delivery, injection delivery, transdermal delivery,and the like.

All U.S. patents and applications; foreign patents and applications;scientific articles; books; and publications mentioned herein are herebyincorporated by reference in their entirety, including any drawings,figures and tables, as though set forth in full.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A general scheme for producing a composition comprising chimericproteins for V_(L) or V_(H) regions of a particular immunoglobulin fromB cells from a patient having B cell mediated pathology.

FIG. 2: Plasmid map of a baculovirus expression vector p2Bac withmultiple cloning sites.

FIGS. 3A, 3B and 3C: DNA sequence of baculovirus expression vector p2Bac(SEQ ID NO:5). The sequence is depicted from 5′ to 3′. The p2Bac vectorcontains the AcNPV polyhedrin gene promoter (nucleotides 1 to 120 of theGenBank accession number X06637 (SEQ ID NO:92)) and the AcMNPV p10promoter (nucleotides 8 to 237 of GenBank accession number A28889 (SEQID NO:93)).

FIG. 4: DNA sequence of the plasmid pTRABac/9F12. This plasmid containsthe genes for the heavy and light (K) chains expressed by the stablehuman cell-line 9F12. This cell line produces a human IgG1/K, antibodyspecific for tetanus toxoid (SEQ ID NO:89). The underlined regionsrepresent sequences encoding mature 9F12 IgG₁ (TTTACCC . . . ) and kappa(ATCGACA . . . ) chains, respectively. The sequence is depicted from 5′to 3′.

FIG. 5A: Plasmid map of recombinant baculovirus expression vectorpTRABacHuLC_(K) HC_(γ1) with IgG_(γ1) constant regions.

FIG. 5B: Plasmid map of recombinant baculovirus expression vectorpTRABacHuLC_(λ) HC_(γ1) with IgG_(γ1) constant regions.

FIG. 6A: DNA sequence of pTRABacHuLC_(K)HC_(γ1) (SEQ ID NO:6). Thesequence is depicted from 5′ to 3′.

FIG. 6B: DNA sequence of pTRABacHuLC_(λ) HC_(γ1) (SEQ ID NO:7). Thesequence is depicted from 5′ to 3′.

FIG. 6C: DNA sequence of pTRABacHuLC_(K) HC_(γ1) following modificationutilizing the kappa stuff primers (SEQ ID NO:90). The sequence isdepicted from 5′ to 3′.

FIG. 6D: DNA sequence of pTRABacHuLC_(λ) HC_(γ1) following modificationutilizing the lambda stuff primers (SEQ ID NO:91). The sequence isdepicted from 5′ to 3′.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The possibility of evoking an immune response that would recognize andeliminate neoplastic cells while sparing normal tissue represents anexciting approach to the treatment of cancer. Inducing such an immuneresponse is assisted-by identifying a unique tumor antigen. B-cellmalignancies express a unique antigen, the immunoglobulin idiotype (Id),on their surface. This antigen contains protein sequences from both thevariable immunoglobulin heavy and light regions (V_(L) and V_(H)). EachB-cell harbors a unique genetic sequence used in the production of theimmunoglobulin idiotype. Consequently, as most B cell malignancies arisefrom the clonal expansion of a single B cell, all cells comprising aB-cell malignancy expresses a unique Id protein. Hence, idiotypicprotein should serve as an ideal target for immune-based therapy of anyB cell malignancy, such as lymphoma or leukemia.

Passive Immunotherapy

Early immunotherapy strategies focused on the use of monoclonalantibodies against tumor-specific idiotype (anti-Id MoAb). This approachresulted in tumor regression and long-lasting remissions in severalpatients with non-Hodgkin's lymphoma. However many patients experiencedeventual relapse (Miller et al., N. Engl. J. Med. 306(9):517-22, 1982;Maloney et al., Blood 80(6):1502-10, 1992; Brown et al., Blood73(3):651-61, 1989; Brown et al., Semin. Oncol 16(3):199-210, 1989;Meeker et al., Blood 65(6):1349-63, 1985.

One difficulty that arose in the studies described above was that cellsof a malignant B or T cell lymphoma could alter their expression oftheir idiotypic immunoglobulins or T cell receptors. Two examples ofthis were described in some of the articles listed above (Cleary et al.,1986, and Meeker et al., 1985). It was also shown that T cell leukemiacells could escape anti-idiotypic antibodies by reducing theirexpression of surface T cell receptor (Maecker et al., J. Immunol.141:2994-3002, 1985), and this was confirmed for B cell leukemias in ananimal model (Stevenson et al., J. Immunol. 130(2):970-3, 1983). Otherstudies demonstrated that that there is idiotypic variation even withina given human B cell lymphoma (Berinstein et al., J. Immunol.144(2):752-8, 1990; Levy S, et al., J. Exp. Med. 168(2):475-89, 1988;).Such mutations appeared responsible for the decreased effectiveness ofthe anti-Id MoAb over time (Berinstein et al., supra; Tao et al., Nature362(6422):755-8, 1993; Chen et al., J. Immunol. 153(10):4775-87, 1994).

One way to avoid this problem is via the generation and use of apolyclonal antisera against the idiotypic protein. Caspar and co-workersstudied the potential of a polyclonal antibody-based therapy in a mousemodel system (Caspar et al., Blood 90:3699-706, 1997). These authorsvaccinated a mouse using idiotypic proteins from a non-Hodgkin'sleukemia patient who had relapsed following successful monoclonalantibody therapy. The resultant polyclonal antibodies recognizedidiotypic proteins from both the original tumor and all variants.Therefore, generation of polyclonal antibody response specific to theidiotype of a B cell lymphoma or leukemia would represent an improvementover monoclonal antibody therapy. Producing sufficient quantities ofprotein to for a vaccination to produce polyclonal antibodies is asignificant burden of this approach.

Active Immunotherapy

Active immunotherapy may avoid the phenomenon of mutational escape seenwith passive immune strategies. Such therapy has the potential togenerate a broader immune response and thereby recognize theheterogeneous tumor cell population that can arise over time. Thedifficulty with active immunotherapy lies in convincing the patient'simmune system to react against a perceived “self antigen” expressed bythe tumor. As with idiotypic protein, many of the antigens expressed bytumors are weak immunogens.

In the instant invention, the unique specificity of the immune systemhas been adapted to treat B cell malignancies. In the instant invention,the DNA sequence encoding the variable region of the idiotypicimmunoglobulins was cloned using primers derived from the 5′ end of eachunique subfamily of light and heavy immunoglobulin chains together witha constant region primer. Typically, this process uses one of severalsuitable cloning techniques such as PCR. These constant region primers,in combination with one for the V_(H) region and one for the V_(L)region, maybe used to clone the variable regions as a first step inproducing a chimeric protein comprising a variable region and a constantregion. Alternatively, techniques such as 5′ RACE may be used. In thecase of one patient described infra, 5′ RACE was used to clone thevariable regions of the heavy and light immunoglobulin chains in orderto produce a chimeric protein. Examples of chimeric proteins include:V_(L)/C_(K), V_(L)/C_(λ), V_(L)/IgG_(γ1), V_(H)/IgG_(γ1), V_(H)/C_(K),and V_(H)/C_(λ). These chimeric proteins are produced in insect cellsusing a baculovirus vector. The chimeric protein thus comprises aportion of a variable region from an immunoglobulin molecule from apatient and also comprises a portion of a constant region from a sourceother than the patient. In preferred embodiments, the heavy and lightchain constant regions are derived from 9F12 cells. However, othersources for immunoglobulin constant region genes may be used. Thesechimeric proteins are predicted to be more efficiently produced thanusing existing systems for producing idiotypic proteins and will beexcellent immunogens for use in vaccination protocols.

The present invention fills the great demand for an effective treatmentfor B cell mediated pathologies and autoimmune diseases. The inventionstake advantage of the unique cell surface antigens present on thesurface of B cells involved in B cell pathologies, and are prepared in apatient-specific manner. Such vaccines provide exquisite selectivity bybeing tailored to the markers unique to the pathogenic B cells found ina given patient.

The novel baculovirus/insect cell expression system has proven effectivefor the efficient production of functional antibodies for immunotherapyfrom any given patient. This baculovirus expression vector was designedsuch that only two custom gene-specific primers were needed to amplifyany pair of antibody variable regions for easy subcloning and expressionas human kappa light chain and IgG_(γ1) heavy chain. The incorporationof heterologous secretary signal sequences, which directed the heavy andlight chains to the secretary pathway, were incorporated for theexpression of large amounts of active immunoglobulin from insect cells.This vector should be useful for the expression of any kappa light chainvariable region (V_(L)) in frame with human kappa constant region andsecreted via the human placental alkaline phosphatase secretary signalsequence; and any heavy chain variable region (V_(H)) in frame with thehuman IgG_(γ1) constant domain led by the honey bee melittin secretarysignal sequence. In other systems, the lambda light chain constantregion replaces the kappa constant region. The chimeric protein is thenexpressed with the V_(L) region in frame with human lambda constantregion and secreted via the human placental alkaline phosphatasesecretary signal sequence, along with any heavy chain variable region(V_(H)) in frame with the human IgG_(γ1) constant domain led by thehoney bee melittin secretary signal sequence. Any monoclonal antibody,mouse or human, either from a monoclonal cell line or identified byphage display cloning, could be easily expressed as whole humanIgG_(γ1)/K or IgG_(γ1)/λ in this vector after two simple subcloningsteps. Additionally, different immunoglobulin types, including IgG_(γ2),IgG_(γ3), IgG_(γ4), IgA, IgA, IgA₁, IgA₂, IgM, IgD, IgE heavy chains, orsegments thereof, could be used in place of IgG_(γ1). Furthermore,besides those signal sequences described supra, the instant inventionmay use other secretory signal sequences such as the endogenoussecretory sequences associated with the immunoglobulin genes derivedfrom a given patient. Additionally, one of skill in the art would beable to select several different primers that could be used equivalentlyin this system to produce equivalent results to amplify any pair ofantibody variable regions for easy subcloning.

In some instances, utilization of the baculovirus system for theexpression of biologically active proteins has been hampered by theinability to efficiently solubilize recombinant proteins withoutexcessive proteolytic degradation. In order to circumvent solubility andproteolysis problems encountered with the expression of recombinantproteins in insect cells, baculovirus transfer vectors were developedfor the efficient secretion of biologically active proteins. Thesevectors that facilitate the secretion of recombinant proteins from hostinsect cells are constructed by inserting functional secretory leadersequences downstream of the polyhedrin promoter. In-frame insertion ofcDNA sequences resulted in the synthesis of proteins containing aheterologous signal sequence which directed the recombinant protein tothe secretory pathway. Human and insect leader sequences were bothtested to maximize secretion of heterologous proteins from insect cells.The human placental alkaline phosphatase signal sequence (SEQ ID NO: 1:MLGPCMLLLLLLLGLRLQLSLG; DNA sequence is SEQ ID NO:2: ATG GTG GGA CCC TGCATG CTG CTG CTG CTG CTG CTG CTA GGC CTG AGG CTA CAG CTC TCC CTG GGC) andthe honeybee melittin signal sequence (SEQ ID NO:3:MKFLVNVALVFMVVYISYIYA; DNA sequence is SEQ ID NO:4: ATG AAA TTC TTA GTCAAC GTT GCA CTA GTT TTT ATG GTC GTG TAC ATT TCT TAC ATC TAT GCG) haveboth proved useful for the secretion of numerous bacterial and humanproteins (Mroczkowski et al., J. Biol. Chem. 269:13522-28, 1994 andTessier et al., Gene 98:177-83, 1991).

To tailor the present invention to a particular patient first requiresidentification and isolation of the genes encoding the unique antigens,and then the means of producing those antigens. This may be accomplishedin a number of different ways available to one of skill in the art. Forexample, a recently developed method that is adapted to the needs of theinstant invention uses a novel baculovirus/insect cell expression systemand was recently developed for the efficient production of functionalantibodies for immunotherapy (see U.S. Provisional Application Ser. No.60/244,722, entitled “Expression Vectors for Production of RecombinantImmunoglobulin”).

Expression of recombinant proteins using the baculovirus system allowsthe production of large quantities of biologically active proteinswithout many of the drawbacks associated with proteins made in bacteria,and also avoids the complications of using mammalian cells. For examplethe immunoglobulin genes from the stable human cell-line 9F12(ATCC#HB8177), which produces a human IgG1/K antibody specific fortetanus toxoid, were cloned into a baculovirus dual promoter expressiontransfer vector. Intact IgG1/K immunoglobulin was produced in insectcells that behaved similarly to the mammalian antibody in SDS-PAGEanalysis and Western blots. The antibody produced by insect cells wasglycosylated. The binding affinities of purified Mab9F12 and purifiedbaculovirus expressed antibody were determined to be identical andproduction levels were determined to be approximately 5-10 μg/ml.

Soluble human immunoglobulin fragments containing specific epitopes ofthe particular variable regions can be produced in insect host cells viagenetic engineering. These soluble recombinant immunoglobulin proteinscontaining patient-derived particular V_(L) and/or V_(H) regions can beused as a therapeutic composition. When administered into the patient,it would specifically induce, in vivo, a cell mediated immune responsefor altering the B cell mediated pathology.

This technology has also been applied towards the rapid identificationand cloning of patient-specific V_(α) and V_(β) genes expressed by a Tcell lymphoma, then expressing these as recombinant K/V_(α) orIgG_(γ1)/V_(β) molecules in insect cells (see U.S. ProvisionalApplication Ser. No. 60/266,133 entitled “Method and Composition forAltering a T Cell Mediated Pathology”). Molecules produced by thismethod were formulated and used to induce anti-idiotypic cell-mediatedimmunity against lymphomas in a patient-specific fashion.

The term “altering” or “alters” refers to the ability of a compound orcomposition of the invention to modulate a B cell mediated pathology. Acompound which alters a B cell pathology may do so by a number ofpotential mechanisms, including raising antibodies directed at thecompound which in turn destroys cells of the B cell pathology, inducingapoptosis in the B cells involved in the pathology, inhibiting furthergrowth and division of cells of the B cell pathology, inducingcell-mediated immunity directed at the cells of the B cell pathology, orotherwise inhibiting the activity of the pathological B cells. The exactmechanism that causes the alteration need not be determined, but onlythat an alteration in the B cell mediated pathology occurs by somemechanism as a consequence of adding the inventive molecules orcompositions.

The term “B cell mediated pathology” or “B cell pathology” refers tothose diseases and conditions that arise from inappropriate replicationor activity of B cells. In preferred embodiments, the B cell mediatedpathology is a B cell lymphoma that results from inappropriatereplication of B cells. B cell lymphomas are difficult to treateffectively with the currently available medical methods. Other types ofB cell pathologies which involve inappropriate replication of B cellsinclude chronic and acute B cell leukemias, multiple myelomas, and somenon-Hodgkin's lymphomas. Other preferred embodiments include a growingnumber of human diseases that have been classified as autoimmunedisease, where the host's own immune system attacks the host's owntissue, such as multiple sclerosis (MS) (Warren and Catz, Mult. Scler.6(5):300-11, 2000), systemic lupus erythematosus (SLE) (Zhang, J. etal., J. Immunol. 166(1):6-10, 2001; Odendahl, M. et al., J. Immunol.165(10):5970-79, 2000), anti-Hu associated paraneoplastic neurologicalsyndromes (Rauer, S. and Kaiser, R., J. Neuroimmunol. 111(1-2):241-44,2000); autoimmune hepatitis (AIH) (Ogawa, S. et al., J. Gastroenterol.Hepatol (1):69-75, 2000). Other candidate autoimmune diseases fortreatment by the present invention include rheumatoid arthritis (RA),myasthenia gravis (MG), autoimmune thyroiditis (Hashimoto'sthyroiditis), Graves' disease, inflammatory bowel disease, autoimmuneuveoretinitis, polymyositis, scleroderma, and certain types of diabetes.The present treatments for these autoimmune diseases do not cure thedisease, but instead only ameliorate the symptoms.

The term “B cell” refers to a cell of the immune system of an organismwhich is involved in the humoral immunity in normal functioning of aorganism (i.e., one that is not experiencing a B cell mediatedpathology). B cells are white blood cells that develop from bone marrowand produce antibodies; they are also known as B lymphocytes. Ingeneral, B cells are cells involved in antibody production in anorganism.

The term “pathology” refers to a state in an organism (e.g., a human)which is recognized as abnormal by members of the medical community. Thepathology to be treated in the present invention is characterized by anabnormality in the function of B cells.

The term “patient” refers to an organism in need of treatment for apathology, or more specifically, a B cell pathology. The term refers toa living subject who has presented at a clinical setting with aparticular symptom or symptoms suggesting the need for treatment with atherapeutic agent. The treatment may either be generally accepted in themedical community or it may be experimental. In preferred embodiments,the patient is a mammal, including animals such as dogs, cats, pigs,cows, sheep, goats, horses, rats, and mice. In further preferredembodiments, the patient is a human. A patient's diagnosis can alterduring the course of disease progression, either spontaneously or duringthe course of a therapeutic regimen or treatment.

An “organism” can be a single cell or multi-cellular. The term includesmammals, and, most preferably, humans. Preferred organisms include mice,as the ability to treat or diagnose mice is often predictive of theability to function in other organisms such as humans. Other preferredorganisms include primates, as the ability to treat or diagnose primatesis often predictive of the ability to function in other organisms suchas humans.

The term “chimeric protein” refers to a protein which comprises a singlepolypeptide chain comprising segments derived from at least twodifferent proteins. The segments of the chimeric protein must be derivedfrom heterologous proteins, that is, all segments of the chimericpolypeptide do not arise from the same protein. The chimeric proteins ofthe present invention include proteins containing portions of the V_(L)or V_(H) region of an immunoglobulin chain, but do not comprise theentire C region of those chains as found in the B cell clone from whichthe V_(L) or V_(H) regions is derived. Furthermore, the V_(L) or V_(H)region may not include the entire variable region, but does includeenough to generate an immune response. Chimeric proteins of the presentinvention may also include proteins in which a segment of the naturallyoccurring protein has been replaced with an equivalent naturally ornon-naturally occurring segment. This includes replacing the IgG₁constant region derived from a patient with the IgG₁ constant regionfrom a different source and would also include immunoglobulin constantregions in which a segment of the protein has been replaced with alinker, segment or domain that is partially or entirely manmade. In allcases, however, the gene for the chimeric protein of the instantinvention will not be the same as the gene for the immunoglobulins whichoccur naturally in the patient. The gene for the chimeric protein willbe distinguishable from naturally occurring protein for one of thefollowing reasons: (1) it will not be the full length immunoglobulingene or cDNA from the patient, (2) it will be a different subtype thanisolated from the patient, or (3) the nucleic acid sequence encoding thepatient's IgG₁ constant region will differ from the IgG₁ gene used inthe expression vector.

The terms “protein,” “polypeptide,” and “peptide” are used hereininterchangeably.

The term “naturally” or “native” refers to a protein as it is isolatedfrom nature. Thus, a naturally occurring protein may refer to a proteinas it is found in nature which is encoded by a gene that has not beenmodified by the use of recombinant techniques. A native protein mayrefer to a protein as it may be found or synthesized in nature. Theseterms may also apply to proteins which are produced by biological systemsuch as the bacculovirus virus system of the present invention or by theculture of cells derived from patients. A native protein may alternatelyrefer to an isolated protein which has not been denatured. The term“native” may also refer to the manner in which polypeptide or protein isfolded, either alone or in combination with other polypeptides, so thatit resembles similar proteins found in nature, or how it is modifiedafter translation (“post-translational modifications”) so that itresembles similar proteins found in nature. A naturally-occurringprotein may be found only in pathological B cells from a single patient,nevertheless, this may be considered a naturally-occurring protein.

The term “segment” or “portion” is used to indicate a polypeptidederived from the amino acid sequence of the proteins used for thechimeric proteins having a length less than the full-length polypeptidefrom which it has been derived. It is understood that such segments mayretain one or more characterizing portions of the native polypeptide.Examples of such retained characteristics include: binding with anantibody specific for the native polypeptide, or an epitope thereof.

The terms “V_(H)” and “V_(L)” refer to the variable regions of thepolypeptide chains of immunoglobulin molecules, or nucleic acidsencoding such polypeptide chains. One skilled in the art realizes themeaning of these terms. The exact sequence of a variable region cannotbe predicted and must be determined by isolating the sequence inquestion. The V_(L) and V_(H) regions isolated from particular patientsare used in the instant invention. The exact sequence of a kappa (K) orlambda (λ) light chain is determined by clonal rearrangements of the Vregions, J regions and Constant region of the light chain locus. (Thekappa and lambda loci are separate and distinct.) The exact sequence ofa heavy chain is determined by clonal rearrangements of the V regions, Dregions, J regions and Constant region of the heavy chain locus.Additional sequence variation in the variable region arises fromimprecision during the recombination process and also is generated bysomatic mutations′subsequent to the end of the recombination process.The terms “V_(H)” and “V_(L)” also refer to portions or segments of theVL and VH regions. A segment of the V_(L) and V_(H) region may alsoinclude all or substantially all of the V region. The term“substantially all” refers to approximately 90% of the entire variableregion, or approximately 80% of the entire variable region. The portionof the V_(L) and V_(H) region present must be sufficient to allow thechimeric molecule to operate in the present invention. The terms “V_(H)”and “V_(L)” also refer to functional derivatives of such polypeptideregions as described infra.

The term “immunoglobulin constant region” refers to all or part of thatportion of immunoglobulin molecules which are not encoded by thevariable regions of immunoglobulins. The term “immunoglobulin constantregion” may also refer to the DNA sequence encoding the immunoglobulinconstant region. The immunoglobulin constant region includes thesegments C_(L), C_(H1), C_(H2), C_(H3), and the Hinge region.Immunoglobulin types include IgGγ1, IgG_(γ2), IgG_(γ3), IgG_(γ1), IgA₁,IgA₂, IgM, IgD, IgE heavy chains, and K or λ light chains or segmentsthereof. Any immunoglobulin constant region segments may be used in theinstant invention, provided that the segment allows the immunoglobulinconstant region to operate for the purposes of the present invention,for example, or the affinity purification of the chimeric molecule, viabinding to Protein G, Protein A, Protein L, or appropriate antibody.Functional derivatives of the immunoglobulin constant region segments,as described infra, may also be used.

The term “immunoglobulin fold” or “immunoglobulin domain” refers to astructural element of the immunoglobulin super family. Theimmunoglobulin domain is a conserved, repeating structural domain ofapproximately 110 amino acids each.

Immunoglobulin domains are found in many protein molecules, includingantibodies, the T cell antigen receptor, cytokine receptors (e.g., theplatelet-derived growth factor receptor with 5 Ig domains), celladhesion molecules (e.g., ICAM-1/CD54), and many others. Twoimmunoglobulin domains are found in each TCR; one in the variable regionand one in the constant region. Two immunoglobulin domains are found inantibody light chains and four are found in IgG heavy chains. Thepresent invention contemplates the replacement of one or two domains ofthe constant region with domains from a different molecule, such as animmunoglobulin molecule, to produce a modified (chimeric) constantregion which may have different properties such as binding to othermolecules.

The terms “IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgA₁, IgA₂, IgM, IgD, IgE” referto classes and subclasses of human immunoglobulins. The terms may referto either the DNA sequences or the amino acid sequences of the proteins.The class and subclass of an immunoglobulin molecule is determined byits heavy chain. IgG and IgD are different classes of immunoglobulins;IgG₁ and IgG₂ are different subclasses of immunoglobulin molecules. Theterm “IgA” may refer to any subclass of IgA molecules. In preferredembodiments, it refers to an IgA₁ molecule. In other preferredembodiments, it refers to an IgA₂ molecule. In some embodiments, theimmunoglobulin heavy chain used may be a chimeric protein that containsamino acids from a second protein.

The term “IgG_(γ1)” refers to the heavy chain associated with the IgG₁class of immunoglobulins. IgG₁ represents approximately 66% of human IgGimmunoglobulins Roitt et al., Immunology, Mosby, St. Louis, pg. 4.2,1993).

The terms “kappa constant region,” “lambda constant region,” “K constantregion,” and “λ constant region” refer to the constant regions of kappa(K) and lambda (λ) light chains that remain constant during thedevelopment of the immune system. The terms may refer to either the DNAsequences or the amino acid sequences of the proteins. In someembodiments, portions of the immunoglobulin light chain may be comprisedin a chimeric protein that contains amino acids from one or more otherproteins.

The term “administering” relates to a method of contacting a compoundwith or into cells or tissues of an organism. The B cell mediatedpathology can be prevented or treated when the cells or tissues of theorganism exist within the organism or outside of the organism. Cellsexisting outside the organism can be maintained or grown in cell culturedishes. For cells harbored within the organism, many techniques exist inthe art to administer compounds, including (but not limited to) oral,parenteral delivery, decimal application, injection, and aerosolapplications.

The B cell mediated pathology can also be prevented or treated byadministering a compound of the invention, or an antibody raised to acompound of the invention, to B cells displaying the characteristics ofa pathology. The effect of administering a compound on organism functioncan then be monitored. The organism is preferably a mouse, rat, rabbit,guinea pig, or goat, more preferably a monkey or ape, and mostpreferably a human.

The term “composition” refers to a mixture that contains the protein ofinterest. In preferred embodiments, the composition may containadditional components, such as adjuvants, stabilizers, excipients, andthe like.

The term “associated with” in reference to the relation of a variableregion to a B cell clone refers to the variable region that is found onthe immunoglobulins produced by a particular B cell clone.

The term “B cell clone” refers to the clonal descendants of a single Bcell. Clonal descendants of B cells express the same idiotype in theproduced antibodies as the parental cell. One skilled in the artrealizes that clonal descendants of a B cell may have undergone somaticmutation within the variable region of the immunoglobulin gene but stillremain part of the B cell clone.

The term “isolating” refers to removing a naturally occurring nucleicacid sequence from its normal cellular environment. Thus, the sequencemay be in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the onlynucleotide chain present, but that it is essentially free (about 90-95%pure at least) of non-nucleotide material naturally associated with it,and thus is distinguished from isolated chromosomes. Also, by the use ofthe term “isolating” in reference to nucleic acid is meant that thespecific DNA or RNA sequence is increased to a significantly higherfraction (2- to 5-fold) of the total DNA or RNA present in the solutionof interest than in the cells from which the sequence was taken. Thiscould be caused by a person by preferential reduction in the amount ofother DNA or RNA present, or by a preferential increase in the amount ofthe specific DNA or RNA sequence, or by a combination of the two.However, it should be noted that enriched does not imply that there areno other DNA or RNA sequences present, just that the relative amount ofthe sequence of interest has been significantly increased. The term“significant” is used to indicate that the level of increase is usefulto the person making such an increase, and generally means an increaserelative to other nucleic acids of about at least 2-fold, morepreferably at least 5- to 10-fold or even more. The term also does notimply that there is no DNA or RNA from other sources. The DNA from othersources may, for example, comprise DNA from a yeast or bacterial genome,or a cloning vector such as pUC19. This term distinguishes fromnaturally occurring events, such as viral infection, or tumor-typegrowths, in which the level of one mRNA may be naturally increasedrelative to other species of mRNA. That is, the term is meant to coveronly those situations in which a person has intervened to elevate theproportion of the desired nucleic acid.

Isolated DNA sequences are relatively more pure than in the naturalenvironment (compared to the natural level this level should be at least2- to 5-fold greater, e.g., in terms of mg/mL). Individual sequencesobtained from PCR may be purified to electrophoretic homogeneity. TheDNA molecules obtained from this PCR reaction could be obtained fromtotal DNA or from total RNA. These DNA sequences are not naturallyoccurring, but rather are preferably obtained via manipulation of apartially purified naturally occurring substance (e.g., messenger RNA(mRNA)). For example, the construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection from the cells carrying the cDNA library. The processwhich includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated.

The term “gene encoding” refers to a sequence of nucleic acids whichcodes for a protein or polypeptide of interest. The nucleic acidsequence may be either a molecule of DNA or RNA. In preferredembodiments, the molecule is a DNA molecule. In other preferredembodiments, the molecule is a RNA molecule. When present as a RNAmolecule, it will comprise sequences which direct the ribosomes of thehost cell to start translation (e.g., a start codon, ATG) and direct theribosomes to end translation (e.g., a stop codon). Between the startcodon and stop codon is an open reading frame (ORF). One skilled in theart is very familiar with the meaning of these terms.

The term “insect cell lines” refers to cell lines derived from insectsand susceptible to infection by the bacculovirus. One skilled in the artis familiar with such cell lines and the techniques needed to utilizethem. Representative examples of insect cell lines include Spodopterafrugiperda (sf9) and Trichoplusia ni (Hi-5) cell lines.

The terms “Trichoplusia ni (High-5) cells” and “Spodoptera frugiperda(sf9) cells” refers to insect cell lines used in combination withbaculovirus expression vectors. One skilled in the art is familiar withthese cell lines and how to obtain them.

The term “inserting” refers to a manipulation of a DNA sequence via theuse of restriction enzymes and ligases whereby the DNA sequence ofinterest, usually encoding the gene of interest, can be incorporatedinto another nucleic acid molecule by digesting both molecules withappropriate restriction enzymes in order to create compatible overlapsand then using a ligase to join the molecules together. One skilled inthe art is very familiar with such manipulations and examples may befound in Sambrook et al. (Sambrook, Fritsch, & Maniatis, “MolecularCloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory,1989), which is hereby incorporated by reference in its entiretyincluding any drawings, figures and tables.

The term “adjuvant” refers to a substance which is provided with theantigen or immunogen of choice, e.g., the protein or polypeptide towhich an immune response is desired, to enhance the immune response whenone attempts to raise an immune response in an animal against theantigen or immunogen of choice. One skilled in the art is familiar withappropriate adjuvants to select and use. Adjuvants approved for humanuse include aluminum salts and MF59 (Singh and O'Hagan, Nature Biotech17:1075-81, 1999). Other adjuvants are being developed (Id.) and may beused in conjunction with the present invention.

The term “keyhole-limpet hemocyanin” or “KLH” refers to a protein whichis isolated from keyhole limpets which is commonly used as a carrierprotein in the immunization process. One skilled in the art is familiarwith the meaning of the term keyhole limpet hemocyanin.

The term “cytokine” refers to a family of growth factors, soluble(glyco)proteins, secreted primarily from leukocytes. Cytokines stimulateboth the humoral and cellular immune responses, as well as theactivation of phagocytic cells. Cytokines are synthesized, stored andtransported by various cell types not only inside of the immune system(lymphokines, interleukins, monokines, tumor necrosis factors,interferons) but also by other cells which are associated with the studyof hematology (colony-stimulating factors), oncology (transforminggrowth factors), and cell biology (peptide growth factors, heat shockand other stress proteins).

Cytokines secreted from lymphocytes are termed lymphokines, while thosesecreted by monocytes or macrophages are referred to as monokines. Manyof the lymphokines are also referred to as interleukins (ILs), sincethey are not only secreted by leukocytes but they are also able toaffect the cellular responses of leukocytes. Specifically, interleukinsare growth factors targeted to cells of hematopoietic origin.

The term “growth factor” refers to a protein that binds receptors on thesurface of a cell and subsequently activates cellular proliferationand/or differentiation. Many growth factors are quite versatile and canact to stimulate cellular division in a wide variety of cell types,while others are specific to a particular cell-type.

The term “chemokine” refers to a group of small proinflammatorycytokines which function as chemoattractants and activators forleukocytes and represent a superfamily of over 30 chemotactic cytokines.They orchestrate the activation and migration of immune system cellsfrom the blood or bone marrow to the site of infection and damagedtissue. Chemokines also play an essential role in the growth andproliferation of primitive stem cells found in bone marrow which in turndevelop into mature immune cells. Chemokines are involved in a widerange of acute and inflammatory diseases and exert their action bybinding to receptors of the seven-transmembrane-helix class.

Chemokines frequently range from 8 to 11 kDa in molecular weights, areactive over a concentration range of 1 to 100 ng/ml, and are produced bya wide variety of cell types. The production of chemokines typically isinduced by exogenous irritants and endogenous mediators such as IL-1,TNF-alpha, and PDGF. The chemokines bind to specific cell surfacereceptors and can be considered second-order cytokines that appear to beless pleiotropic than first-order proinflammatory cytokines because theyare not potent inducers of other cytokines and exhibit more specializedfunctions in inflammation and repair.

The term “granulocyte-macrophage colony-stimulating factor” or “GM-CSF”refers to a small (less than 20 kDa) secreted protein. It binds tospecific cell surface receptors and functions as species-specificstimulator of bone marrow cells. It stimulates the growth anddifferentiation of several hematopoietic cell lineages includingdendritic cells, granulocytes, macrophages, eosinophils, anderythrocytes. In particular, this cytokine also plays a role in shapingcellular immunity by augmenting T-cell proliferation (Santoli et al., J.Immunol. 141(2):519-26, 1988), increasing expression of adhesionmolecules on granulocytes and monocytes (Young et al., J. Immunol.145(2):607-15, 1990; Grabstein et al., Science 232(4749):506-08, 1986),and by augmenting antigen presentation (Morrissey et al., J. Immunol139(4):I.1.13-9, 1986; Heufler et al., J. Exp. Med. 167(2):700-05, 1988;Smith et al., J. Immunol. 144(5):1777-82, 1990).

The term “monocyte chemotactic protein-3” or “MCP-3” refers a chemokineprimarily produced by monocytes. MCP-3 has a wide spectrum ofchemotactic activity and attracts monocytes, dendritic cells,lymphocytes, natural killer cells, eosinophils, basophils, andneutrophils. The cDNA was cloned in 1993 by Minty et al., Eur CytokineNetw 4(2):99-110, 1993, and Opdenakker et al., Biochem Biophys ResCommun., 191(2):535-42, 1993. Its properties have been recently reviewedby Proost et al., J. Leukoc Biol. 59(1):67-74, 1996.

The term “expression vector” refers to a recombinant DNA construct whichis designed to express a selected gene of interest, usually a protein,when properly inserted into the expression vector. One skilled in theart understands the term. Expression vectors commonly include a promotorat the 5′ end of the site where the gene of interest is inserted and aterminator region at 3′ end of the site. Frequently the gene of interestis inserted into the appropriate site by means of selected restrictionenzyme cleavage sites. The term “expression vector” also refers to a DNAconstruct such as described above into which the gene of interestencoding the product of interest has already been inserted.

The term “baculovirus expression vector” refers to a DNA construct whichis designed to express a selected gene when used in the baculovirussystem. Any of the potential baculoviruses or expression vectorsdesigned to function in the baculovirus system may be used in theinstant invention. In a similar fashion, the term “expression vector” isa genus which encompasses the particular embodiment of baculovirusexpression vectors, but “expression vectors” may function in cells andcell lines aside from, or in addition to, insect cell lines.

The term “allow the expression of” refers to placing an expressionvector into an environment in which the gene of interest will beexpressed. This commonly means inserting the expression vector into anappropriate cell type where the promotor and other regions necessary forgene expression will be recognized by the host cell's components andwill cause the expression of the gene of interest. The expressionnormally consists of two steps: transcription and translation.Expression can also be conducted in vitro using components derived fromcells. One skilled in the art is familiar with these techniques, andsuch techniques are set forth in Sambrook et al. (Sambrook, Fritsch, &Maniatis, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold SpringHarbor Laboratory, 1989). In, the preferred embodiment, the expressedproduct is a protein or polypeptide. In other, preferred embodiments,the expressed product is V_(H)/IgG_(γ1), V_(L)/C_(K), V_(L)/C_(λ), orV_(L)/IgG_(γ1).

The term “secretory signal sequence” refers to a peptide sequence. Whenthis sequence is translated in frame as a peptide attached to theamino-terminal end of a polypeptide of choice, the secretory signalsequence will cause the secretion of the polypeptide of choice byinteracting with the machinery of the host cell. As part of thesecretory process, this secretory signal sequence will be cleaved off,leaving only the polypeptide of interest after it has been exported. Inpreferred embodiments, the honey bee melittin secretory signal sequenceis employed. In other preferred embodiments, the human placentalalkaline phosphatase secretory signal sequence is employed. The presentinvention is not limited by these secretory signal sequences and otherswell known to those skilled in the art may be substituted in place of,and in addition to, these. The term “secretory signal sequence” alsorefers to a nucleic acid sequence encoding the secretory peptide.

The term “ELISA” refers to “Enzyme-Linked ImmunoSorbent Assay” in whichthe presence or concentration of a protein is determined by its bindingto the plastic well of an ELISA plate followed by its subsequentdetection by antibodies specific for the protein to be quantified ordetected.

The term “promoter controls” refers to an arrangement of DNA in anexpression vector in which a promoter is placed 5′ to a gene of interestand causes the transcription of the DNA sequence into an mRNA molecule.This mRNA molecule is then translated by the host cell's machinery. Oneskilled in the art is very familiar with the meaning of this term.

The terms “protein A,” “protein G,” and “protein L” refer to specificbacterial proteins which are capable of specifically bindingimmunoglobulin molecules without interacting with an antigen bindingsite. Protein A is a polypeptide isolated from Staphylococcus aureusthat binds the Fc region of immunoglobulin molecules. Protein G is abacterial cell wall protein with affinity for immunoglobulin G (IgG),which has been isolated from a human group G streptococcal strain(G148). Protein L is an immunoglobulin light chain-binding proteinexpressed by some strains of the anaerobic bacterial speciesPeptostreptococcus magnus.

The term “B cell lymphoma” refers to a cancer that arises in cells ofthe lymphatic system from B cells. B cells are white blood cells thatdevelop from bone marrow and produce antibodies. They are also known asB lymphocytes.

The term “refractory low grade B cell lymphoma” refers to a low grade Bcell alymphoma that has not responded to treatment. The term “low gradeB cell lymphoma” refers to a lymphoma that tends to grow and spreadslowly, including follicular small cleaved cell lymphoma. Also calledindolent lymphomas due to their slow growth.

The term “follicular B cell lymphoma” refers to a type of non-Hodgkin'slymphoma. It is an indolent (slow-growing) type of lymphoma.

Further definitions and characterizations of low-grade lymphomas can befound on the Internet athttp://rituxan.com/professional/clinical_-information/class/index.html.

The term “isolating” as refers to a protein or polypeptide, refers toremoving a naturally occurring polypeptide or protein from its normalcellular environment or refers to removing a polypeptide or proteinsynthesized in an expression system (such as the baculovirus systemdescribed herein) from the other components of the expression system.Thus, the polypeptide sequence may be in a cell-free solution or placedin a different cellular environment. The term does not imply that thepolypeptide sequence is the only amino acid chain present, but that itis essentially free (about 90-95% pure at least) of non-amino acid-basedmaterial naturally associated with it.

By the use of the term “enriched” in reference to a polypeptide is meantthat the specific amino acid sequence constitutes a significantly higherfraction (2- to 5-fold) of the total amino acid sequences present in thecells or solution of interest than in normal or diseased cells or in thecells from which, the sequence was taken. This could be caused by aperson by preferential reduction in the amount of other amino acidsequences present, or by a preferential increase in the amount of thespecific amino acid sequence of interest, or by a combination of thetwo. However, it should be noted that enriched does not imply that thereare no other amino acid sequences present, just that the relative amountof the sequence of interest has been significantly increased. The termsignificant here is used to indicate that the level of increase isuseful to the person making such an increase, and generally means anincrease relative to other amino acid sequences of about at least2-fold, more preferably at least 5- to 10-fold or even more. The termalso does not imply that there is no amino acid sequence from othersources. The other source of amino acid sequences may, for example,comprise amino acid sequence encoded by a yeast or bacterial genome, ora cloning vector such as pUC19. In preferred embodiments, the amino acidsequence is a chimeric protein as described above. The term is meant tocover only those situations in which man has intervened to increase theproportion of the desired amino acid sequence.

It is also advantageous for some purposes that an amino acid sequence bein purified form. The term “purified” in reference to a polypeptide doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment. Compared to the natural levelthis level should be at least 2- to 5-fold greater (e.g., in terms ofmg/mL). Purification of at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. The substance is preferably free ofcontamination at a functionally significant level, for example 90%, 95%,or 99% pure.

The term “operatively linked” refers to an arrangement of DNA in which acontrolling region, such as a promoter- or enhancer, is attached to aconnected DNA gene of interest so as to bring about its transcription,and hence allowing its translation. The term “operatively linked” mayalso refer to a DNA sequence encoding a processing signal, such as asecretory signal sequence, connected to a gene encoding a polypeptide toform a single open reading frame. Following transcription andtranslation, the secretory signal sequence has the potential to bringabout the export of the translated polypeptide. One skilled in the artis familiar with-the meaning of this term.

Functional Derivatives of Useful Chimeric Proteins

Also provided herein are functional derivatives of a polypeptide ornucleic acid of the invention. By “functional derivative” is meant a“chemical derivative,” “fragment,” or “variant,” of the polypeptide ornucleic acid of the invention, as these terms are defined below. Afunctional derivative retains at least a portion of the function of theprotein, for example, reactivity with an antibody specific for theprotein or binding activity mediated through noncatalytic domains, whichpermits its utility in accordance with the present invention. It is wellknown in the art that due to the degeneracy of the genetic code numerousdifferent nucleic acid sequences can code for the same amino acidsequence. Equally, it is also well known in the art that conservativechanges in amino acid can be made to arrive at a protein or polypeptidethat retains the functionality of the original. In both cases, allpermutations are intended to be covered by this disclosure.

Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons that specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan, the known aminoacids can be coded for by more than one codon. Thus, portions or all ofthe genes of the invention could be synthesized to give a nucleic acidsequence significantly different from a sequence that is found innature. The encoded amino acid sequence thereof would, however, bepreserved.

A “chemical derivative” of the complex contains additional chemicalmoieties not normally a part of the protein. Covalent modifications ofthe protein or peptides are included within the scope of this invention.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the peptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues, as described below. It may also consist of attachingcarbohydrates to the protein in addition to the normal carbohydratesattached by the bacculovirus expression system of the invention.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect or reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing primary amine containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineα-amino group.

Tyrosyl residues are well-known targets of modification for introductionof spectral labels by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form 0-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimide (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethyipentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutarnyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions; Either form ofthese residues falls within the scope of this invention.

Other modifications include the in vitro glycosylation of polypeptidesor proteins.

Derivatization with bifunctional agents is useful, for example, forcross-linking the component peptides of the protein to each other or toother proteins in a complex to a water-insoluble support matrix or toother macromolecular carriers. Commonly used cross-linking agentsinclude, for example, 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such as methyl-3-[p-azidophenyl]dithiolpropioimidateyield photoactivatable intermediates that are capable of formingcrosslinks in the presence of light. Alternatively, reactivewater-insoluble matrices such as cyanogen bromide-activatedcarbohydrates and the reactive substrates described in U.S. Pat. Nos.3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 areemployed for protein immobilization.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86, 1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility,absorption, biological half life, and the like. The moieties mayalternatively eliminate or attenuate any undesirable side effect of theprotein complex and the like. Moieties capable of mediating such effectsare disclosed, for example, in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990).

A functional derivative of a protein with deleted, inserted and/orsubstituted amino acid residues may be prepared using standardtechniques well-known to those of ordinary skill in the art. Forexample, the modified components of the functional derivatives may beproduced using site-directed mutagenesis techniques (as exemplified byAdelman et al., DNA 2:183, 1983) wherein nucleotides in the DNA codingthe sequence are modified such that a modified coding sequence ismodified, and thereafter expressing this recombinant DNA in aprokaryotic or eukaryotic host cell, using techniques such as thosedescribed above. Alternatively, proteins with amino acid deletions,insertions and/or substitutions may be conveniently prepared by directchemical synthesis, using methods well-known in the art. The functionalderivatives of the proteins typically exhibit the same qualitativebiological activity as the native proteins.

Uses of the Chimeric Proteins of the Invention

Other aspects of the invention relate to uses for the instant chimericproteins. Preferred uses include pharmaceutical and veterinaryapplications, wherein an effective amount of chimeric protein accordingto the invention (preferably in a composition according hereto) isadministered to a patient. In this way, the chimeric protein contactscells of the patient, which contacting thereafter elicits the desiredbiological response. Methods for using the instant chimeric proteinsinclude methods of eliciting an immune response in an organism, methodsof raising antibodies (B cell immune response) in an organism, methodsof inducing a T cell immune response by an organism, and methods fortreating B cell pathologies. The invention also includes methods fortreatment of subjects in order to increase the immune response capableof altering a B cell pathology by administering a chimeric protein ofthe invention.

Typically, such methods are accomplished by delivering to the organisman effective amount of a chimeric protein according to the invention.“Effective amount” refers to an amount that results in the desiredbiological response being elicited. What constitutes such an amount willvary, and depends on a variety of factors, including the particularchimeric protein, the desired biological response to be elicited, theformulation of the chimeric protein, the age, weight, gender, and healthof the organism to be treated, the dosage regimen, the condition ordisease to be treated or prevented, etc. Organisms to which the instantchimeric proteins and compositions may be administered include mammals,preferably a mammal selected from the group consisting of a bovine,canine, equine, feline, ovine, porcine, and primate animal. Particularlypreferred organisms are humans.

The compounds described herein can be administered to a human patientper se, or in pharmaceutical compositions where it is mixed with otheractive ingredients, as in combination therapy, or suitable carriers orexcipient(s). Techniques for formulation and administration of thecompounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition.

1. Routes of Administration.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intranasal, or intraocular injections. One of skill inthe art will understand the various modifications that would be made toadapt the composition to a particular route of administration.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compounddirectly-into a solid tumor, often in a depot or sustained releaseformulation.

2. Composition/Formulation.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Suitable carriers includeexcipients such as, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for compositions may be employed.Liposomes and emulsions are well known examples of delivery vehicles orcarriers for hydrophobic drugs. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. Additionally, the compounds may be delivered using asustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Varioussustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Many of the compounds of the invention may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms.

3. Effective Dosage.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions where the active ingredients are contained in anamount effective to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount of compound effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g.,Fingl et al., “The Pharmacological Basis of Therapeutics,” Ch. 1 p. 1,1975).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain therequired effect, or minimal effective concentration (MEC). The MEC willvary for each compound. Dosages necessary to achieve the MEC will dependon individual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

4. Packaging.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the polynucleotide for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition. Suitable conditions indicated on the labelmay include treatment of a tumor, treatment of rheumatoid arthritis,treatment of diabetes, and the like.

EXAMPLES

In the following description, reference will be made to variousmethodologies known to those skilled in the art of immunology, cellbiology, and molecular biology. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full.

1. Tissue Processing for Non-Hodgkin's Lymphoma Idiotype

(ID) Identification and Cloning:

Tumor samples from a peripheral lymph node were biopsied as clinicallyindicated under sterile conditions and used to generate patientidiotype-specific recombinant chimeric immunoglobulin proteins.Remaining lymph node biopsy material was stored in liquid nitrogen intissue cell bank for future use.

a. Cell Isolation: Single cell suspensions of patient lymph nodebiopsies were obtained by forcing the biopsied lymphoma tissue through adisposable 0.38 mm steel mesh screen while submerged in sterile PBS. Thedispersed cells were washed twice in PBS, then resuspended and counted.A 10% fraction of the cells were processed for total RNA extraction andthe remaining cells were archived in liquid nitrogen followingresuspension in RPMI 1640 tissue culture media containing 30% (v/v)fetal bovine serum and 10% (v/v) DMSO. All processing of clinicalsamples was performed in a biological safety cabinet.

b. Total RNA Preparation: Total RNA from homogenized lymph node cellswas isolated using RNeasy Kit (Qiagen) as per manufacturer'sinstruction. Total RNA was quantitated by spectrophotometry.

c. cDNA Synthesis: Approximately 2.0 μg total RNA was used as templatefor first strand cDNA synthesis using the SuperScript PreamplificationSystem (GIBCO-BRL) according to manufacturer's recommendation. Oligo(dT) provided with the kit was used to prime the cDNA.

d. PCR Amplification of Genes Encoding Lymphoma Heavy and Light Chains:Both heavy and light chains from the lymphoma-specific immunoglobulinswere identified as follows. Aliquots of the single stranded lymphomacDNA were combined with a series of V_(H) and V_(L) leadersequence-specific oligonucleotide sense primers representing all knownV_(H), V_(K), and V₂, subfamilies as listed in Table 1, paired with IgM,IgG, IgA, or Ig_(K) and Ig_(λ) constant region specific antisenseprimers. These samples were then amplified by PCR and analyzed byagarose gel electrophoresis.

Parallel reactions were conducted using cDNA prepared from the patient'speripheral blood lymphocytes. A comparison of PCR products generated byeach pair of primers derived from samples containing normal PBL or lymphnode biopsy cDNA would lead to the identification of the candidate tumorspecific V_(H) and V_(K) or V_(λ) subfamily over-represented in thelymphoma, and the isotype of the heavy and light chains. Candidate tumorV region gene products were then excised, and their nucleic acidsequence was determined to assess clonality. For each patient, twoindependent analyses were performed from starting cellular fractions.

One microliter of the cDNA reaction (representing 5% of the total cDNAreaction volume) was amplified for 35 cycles in 50 μl volume using theHotStarTaq Master Mix Kit (Qiagen). Cycling conditions: 95° C. 15 min,65° C. 4 min, 72° C. 1 min, followed by 94° C. 1 min, 61° C. 30 sec, 72°C. 1 min for 34 cycles; and a final extension step at 72° C. for 7 min.A 10 μl aliquot of each reaction is analyzed by electrophoresis on a 1%agarose gel with ethidium bromide.

TABLE 1 Primer Sequences Used for Amplification of Lymphoma Heavy and Light Chains ′(GA)′ means either a G or an A, ′(TC)′means either a T or a C. PRIMER NAME PRIMER SEQUENCE (5′ 3′) 491 V_(H1)L TCACCATGGACTGGACCTGGAG SEQ ID NO: 38 492 V_(H2)L.1ACCATGGACATACTTTGTTCCACGC SEQ ID NO: 39 493 V_(H2)L.2ACCATGGACACACTTTGCTCCACGC SEQ ID NO: 40 494 V_(H3)L.1ACCATGGAGTTTGGGCTGAGCTG SEQ ID NO: 41 495 V_(H3)L.2ACCATGGAACTGGGGCTCCGCTG SEQ ID NO: 42 496 V_(H4)LAAGAACATGAAACACCTGTGGTTCTTC SEQ ID NO: 43 497 V_(H5)LATCATGGGGTCAACCGCCATCCT SEQ ID NO: 44 498 V_(H6)LACAATGTCTGTCTCCTTCCTCATC SEQ ID NO: 45 516 V_(K)1L ACATGAGGGTCCCCGCTCAGCSEQ ID NO: 46 517 V_(K)2L TCAGCTCCTGGGGCTGCTAATG SEQ ID NO: 47515 V_(K)3L CTTCCTCCTGCTACTCTGGCTC SEQ ID NO: 48 518 V_(K)4LGCAGACCCAGGTCTTCATTTCTC SEQ ID NO: 49 519 V_(K)5L CCAGGTTCACCTCCTCAGCTTCSEQ ID NO: 50 520 V_(K)6L GGTTTCTGCTGCTCTGGGTTCC SEQ ID NO: 51522 V_(λ1)L TCACTG(TC)(GA)CAGGGTCCTGGGC SEQ ID NO: 52 523 V_(λ2)LACTCAGG(GA)CACAGG(GA)TCCTGG SEQ ID NO: 53 524 V_(λ3).1TTGCTTACTGCACAGGATCCGTG SEQ ID NO: 54 525 V_(λ3)L.2 CTTGCTCACTTTACAGGTTCTGTG SEQ ID NO: 55 526 V_(λ3)L.3 CTCACTCTTTGCATAGGTTCTGTG SEQ ID NO: 56 527 V_(λ3)L.4TCAACCTCTACACAGGCTCTATTG SEQ ID NO: 57 528 V_(λ3)L.5CTCACTCTCTGCACAG(GT)CTCTG(AT)G SEQ ID NO: 58 529 V_(λ4).L1CATTTTCTCCACAGGTCTCTGTGC SEQ ID NO: 59 530 V_(λ4)L.2CCTCCACTG(GC)ACAGGGTCTCTC SEQ ID NO: 60 531 V_(λ5)LCTCTCACTGCACAGGTTCCCTC SEQ ID NO: 61 532 V_(λ6)L CGCTCACTGCACAGGTTCTTGGSEQ ID NO: 62 533 V_(λ7)L CTTGCTGCCCAGGGTCCAATTC SEQ ID NO: 63534 V_(λ8)L TGCTTATGGATCAGGAGTGGATTC SEQ ID NO: 64 535 V_(λ9)LCAGTCTCCTCACAGGGTCCCTC SEQ ID NO: 65 536 V_(λ10)LTCACTCACTCTGCAGTGTCAGTG SEQ ID NO: 66 IgG Constant-ECTGAGTTCCACGACACCGTCAC SEQ ID NO: 69 IgM Constant-EGGGAATTCTCACAGGAGACGAGG SEQ ID NO: 70 C_(K)-E TTGGAGGGCGTTATCCACCTTCSEQ ID NO: 71 C_(λ)-E GAAGTCACTTATGAGACACACCAG SEQ ID NO: 72IgG Constant-I GGAAGTAGTCCTTGACCAGGCAG SEQ ID NO: 73 IgM Constant-IGGGAAAAGGGTTGGGCCCGATGCAC SEQ ID NO: 74 C_(K)-IGGGAAAAGGGTTGGGCCCGATGCAC SEQ ID NO: 75 C_(λ)-IGGAACAGAGTGACACTGGGTGCAGCCTTGGGCTG SEQ ID NO: 76 Cλ DownstreamTGCCGTCGGCAGGAGGTATTTCATTATGACTG TCTCCTTGCTATTATGAACATTCTGTAGGGG-C CASEQ ID NO: 77 Cλ-5′ GTCAGCCCAAGGCTGCACCCAGTGTCACTCTGTTCC SEQ ID NO: 78Cλ-3′ CGTATCAAGCTTTTACTATGAACATTCTGTAGGGGCCAC SEQ ID NO: 79 λ-stuff 1CCTTTGATAACACCCA SEQ ID NO: 80 λ-stuff 1′ GTGTTATCAAAGG SEQ ID NO: 81γ1-stuff 1 5′CTAGTTTGATAAGGGCC3′ SEQ ID NO: 82 γ1-stuff 1′ 5′CTTATCAAA3′SEQ ID NO: 83 K-Stuff 1 5′-CCTTTGATAACACCAA3′ SEQ ID NO: 84 K-Stuff 1′5′--3′ SEQ ID NO: 85

e. Cloning and Sequencing of PCR Products: PCR products from reactionsdetermined-to contain the tumor specific variable sequences for heavyand light chains were cloned directly into plasmid pCR2.1-tOPO as permanufacturer's recommendations, and introduced into Top 10 competent E.coli cells (Invitrogen). Twenty four miniprep DNA plasmids were preparedfrom carbenicillin resistant bacterial colonies using the QIAprep SpinMiniprep Kit (Qiagen), and quantitated by spectrophotometry. Two hundredng of each plasmid was sequenced using the Cy5/Cy5.5 Dye Primer CycleSequencing Kit (Visible Genetics). Following the completion of thesequencing reactions, samples were electrophoresed on the OpenGeneAutomated DNA Sequencing System and the data was processed withGeneObjects software package (Visible Genetics). Additional analysisincluding sequence alignments were performed using the SEQUENCHERVersion 4.1.2 DNA analysis software. (GENE Codes Corp.). A V-regionderived sequence could be considered tumor specific if it was present in75% of the samples, for example, if 18 or greater of the 24 form aconsensus group when analyzed using the above software utilizing thedefault parameters. Two independent biopsy samples would be comparedwhen available.

f. cDNA Synthesis and Generation of 5′ RACE Products: Due to theoccurrence of mutations in the V_(H) and V_(L) sequences, it is notpossible at times to identify tumor-specific immunoglobulinrearrangements. As an alternative to the sequence-specific PCR strategysupra, one can employ a 5′ RACE PCR strategy to identify tumor specificimmunoglobulin (Ig) rearrangements. All steps for first strand cDNAsynthesis to the generation of Ig specific PCR products are performedaccording to manufacturer's directions (5′ RACE system for RapidAmplification of cDNA Ends, version 2.0, Gibco BRL), with slightmodification. Approximately 2.5 μg of total RNA is used as template foreach first strand cDNA synthesis in the presence of specific antisenseprimers complimentary to the immunoglobulin heavy and the light chains'constant region utilized by the B cell population of interest (SEQ IDNO:69 for IgG, SEQ ID NO:70 for IgM, SEQ ID NO:71 for C_(K), and SEQ IDNO:72 for C_(λ)). The cDNA reactions are purified over GlassMAX spincartridges, generating a final volume of 50 μl each. A 10 μl aliquot ofeach purified cDNA is oligo(dC) tailed with terminal deoxynucleotidyltransferase in a 25 μl volume, generating the templates to be used forsubsequent PCR reactions. The PCR set up utilizes an upstream primercontaining a poly(G) track provided by the manufacturer and an Igspecific antisense primer interior to that used for cDNA first strandsynthesis (SEQ ID NO:73 for IgG, SEQ ID NO:74 for IgM, SEQ ID NO:75 forC_(K), and SEQ ID NO:76 for C_(λ)). Five μl of template is amplified ina 50 .μl volume as follows: 95° C. for 15 min, 55° C. for 4 min, 72° C.for 1 min, followed by 94° C. for 1 min, 55° C. for 30 sec, 72° C. for 1min for 34 cycles, and a final extension step at 72° C. for 7 min. Thefinal PCR products are separated by electrophoresis on a 1% agarose gelwith ethidium bromide and the band of the appropriate size(.about.500-600 bp) is isolated are cloned into the pCR2.1-TOPO plasmidas described in 1e, supra.

2. Construction of Baculovirus Expression Vectors pTRABacHuLC_(K)HC_(γ1)and pTRABacHuLC_(λ)HC.sub-γ1.

a. Cloning of Secretory Signal Sequences into p2Bac: The base vector forthe pTRABacHuLC_(K)HC_(γ)1 and pTRABacHuLC_(λ)HC_(γ1) constructs wasp2Bac (FIG. 2, SEQ ID NO:5, Invitrogen, Carlsbad, Calif.). Two secretorysignal sequences were cloned into this base vector, and the firstintermediate baculovirus expression vector p2BacM was created. Ingeneral, the vector p2Bac was first modified utilizing complimentaryoligonucleotides encoding the amino terminal domain of the honey beemelittin secretory signal sequence positioned to be undertranscriptional control of the baculoviral AcNPV P10 promoter. Formelittin sequence cloning, 2 μg p2Bac was digested with Not I and Spe Ifor 4 hours at 37° C. The linear vector was purified followingelectrophoresis through a 1% agarose gel using Qiaex II resin (Qiagen,Chatsworth, Calif.). The purified DNA was then eluted with 50 μl waterand the DNA concentration was determined. One fig each of primers Me1S/N(SEQ ID NO:15) and Me1N/S (SEQ ID NO: 16) were mixed in 10 μl digestionbuffer M (Roche Molecular Biochemicals, Indianapolis, Ind.), and heatedto 70° C. for 5 min, then cooled to room temperature to annealcomplimentary primers. Ten percent of the annealed primers was digestedin 20 μl reaction with Not I and Spe I for 4 hours at 37° C., and thedigested primers were purified following electrophoresis through a 15%polyacrylamide gel with Qiaex H resin, and the concentration of the DNAfor annealed primers was determined. The DNAs of p2Bac vector andannealed melittin fragment were ligated at 1:10 vector to insert ratio.The ligation product was transformed using competent XL1-Blue E. coli(Stratagene, San Diego, Calif.) and plated on a LB-carbenicillin agarplate for overnight growing at 37° C. Miniprep colonies were prepared bystandard protocols, and the plasmids were sequenced to check theconstruction. The resulting vector p2BacM contained the melittinsecretory signal sequence.

The p2BacM vector was further modified similarly to encode for the aminoterminal domain of the human placental alkaline phosphatase secretorysignal sequence under transcriptional control of the AcNPV polyhedronpromoter, creating a second intermediate baculovirus expression vectorp2BacMA. The procedure used to introduce the alkaline phosphatasesequence was generally cloned as follows: 2 μg p2BacM plasmid wasdigested with Bam HI and Eco RI, the linear vector was gel purified fromagarose gel with Qiaex II resin and eluted in 50 H1 water. The DNAconcentration of the vector was determined. One jig each of primersAPB/E (SEQ ID NO: 17) and APE/B (SEQ ID NO: 18) were mixed in 10 μldigestion buffer M, and heated to 70° C. for 5 min and then cooled downto room temperature to anneal complimentary primers. Ten percent of theannealed primers was digested in a 20 μl reaction with Bam HI and Eco RIfor 4 hours at 37° C. The digested primers were then purified from 15%polyacrylamide gel with Qiaex II resin. The DNA concentration of thedigested primers was also determined. The linear p2BacM vector andalkaline phosphatase fragment were then ligated at 1:10 vector to insertratio, and the ligation product was transformed using competent XL1-BlueE. coli and plated on a LB-carbenicillin agar plate for overnightgrowing at 37° C. Miniprep colonies were prepared and the plasmids weresequenced to check the construction. The resulting intermediate vectorp2BacMA would contain a secretory signal sequence for a human placentalalkaline phosphatase. The p2BacMA plasmid was further transformed intoSCS-110 E. coli strain (Stratagene) lacking dcm methylase activity forsubsequent cloning of the K constant region into methyl-sensitive Stu Isite.

b. Amplification and Cloning of Constant Regions of IFG_(γ1) and LightChains: The human kappa (K) constant and the human IgG_(γ1) constantdomains of human monoclonal antibody 9F12 were PCR amplified from RNAextracted from the human cell line 9F12 (ATCC#HB8177). The K constantregion was cloned behind the alkaline phosphatase signal sequence. TheIgG_(γ1) constant region was inserted downstream from the melittinsecretory signal sequence thus creating the vector(pTRABacHuLC_(K)HC_(γ1), FIG. 5 a). A vector containing the human lambda(λ) light chain constant region (pTRABacHuLC_(λ)HC_(γ1), FIG. 5 b) wasproduced by replacing the K light chain constant region with a λ lightchain constant region. The light chains were isolated by RT-PCR from achronic lymphocytic leukemia cellular RNA preparation. The detaileddescription of the cloning procedures are as follows.

c. Amplification of 9F12 K and IgGγ1 constant region fragments: TotalRNA from 9F12 cells (ATCC#HB8177) was extracted using the RNeasy Kit(Qiagen) as per the manufacturer's instruction. A single stranded cDNAwas synthesized using SuperScript reverse transcriptase (GIBCO BRL,Rockville, Md.) with oligo(dT) primers. One twentieth of the synthesizedsingle strand cDNA was amplified in 100 μl PCR reactions with ExpandHigh Fidelity Taq (Roche) using K and IgG_(γ1) specific oligonucleotides(SEQ ID NO:21 plus SEQ ID NO:22 and SEQ ID NO:19 plus SEQ ID NO:20,respectively). The fragments from amplified 9F12 immunoglobulin werepurified from 1.5% SeaKem agarose with Qiaex II resin and eluted with 50μl water. The DNA concentrations for the fragments were determined. Thepurified 9F12 immunoglobulin fragments were ligated separately into theTA-II (Invitrogen) PCR cloning vector. The ligation products weretransformed using competent XL1-Blue E. coli and plated on aLB-carbenicillin agar plate for overnight growing at 37° C. Miniprepcolonies were prepared and the plasmid DNA was sequenced.

d. Insertion of the 9F12 K Constant Region into the Expression Vector:For K constant domain, 5 μg plasmid DNA containing a K constant regionand 2 μg of DNA for the vector p2BacMA purified from SCS 110 E. coliwere digested with Stu I and Hind III. A 320 bp fragment containing Kconstant region and a 7.1 kb fragment containing p2BacMA vector were gelpurified with Quiex II and eluted in 50 μl water. The DNA concentrationsfor both fragments were determined. The purified fragments were thenligated with Rapid Ligation Kit (Roche). The ligation products weretransformed using competent XL1-Blue E. coli and plated on aLB-carbenicillin agar plate for overnight growing at 37° C. Miniprepbacterial colonies were prepared and the recombinant DNA was sequencedto verify proper K constant region insertion. The resulting plasmidvector was pTRABacLC_(K).

e. Addition of the IgG_(γ1) Constant Domain to the Vector: The IgG_(γ1)constant domain was added to the vector by first digesting 5 μg ofplasmid DNA containing IgG_(γ1) constant region and 2 μg plasmid DNA forthe vector pTRABacLC_(K) with Spe I and Xba I. A 1 kb fragment ofIgG_(γ1) constant region and a 7.4 kb fragment of pTkAacLC_(K) vectorwere gel purified from agarose plugs with Quiex II and eluted in 50 μlwater. The DNA concentrations for both fragments were determined. Thepurified fragments were then ligated with Rapid Ligation Kit (Roche).The ligation products were transformed using competent XL1-Blue E. coliand plated on a LB-carbenicillin agar plate for overnight growing at 37°C. Miniprep colonies were prepared and the ligation and orientation ofthe IgG_(γ1) insertion were determined by restriction analysis andsequencing of the restriction sites. The resulting recombinant vectorwas pTRABacHuLC_(K)HC_(γ1).

This plasmid, pTRABacHuLC_(K)HC_(γ1) was further refined to addtranslational stop codons between the melittin secretory sequence, andthe C_(γ1) region sequence and the alkaline phosphatase secretorysequence and the C_(K) region sequence, respectively. To accomplishthese modifications, the pTRABacHuLC_(K)HC_(γ1) vector was linearizedfollowing digestion with Spe I.+Apa I. The linearized vector was thenligated with annealed complimentary primers γ1-stuff 1 (SEQ ID NO:82)and .gamma.1-stuff 1′ (SEQ ID NO:83) to introduce the in-frame stopcodons. The vector resulting from this modification was subsequentlylinearized following digestion with Stu I (AGGCCT)+Dra III (CACnnnGTG)and then ligated with annealed complimentary primers K-stuff 1 (SEQ IDNo. 84) and K-stuff 1′ (SEQ ID NO:81) to introduce the in-frame stopcodons. The net effect of these modifications are indicated in thesequences shown in FIGS. 6C & 6D, respectively. (The added sequences arehighlighted by a double underline and bold.) f. Addition of the λConstant Region to the Vectors: Total RNA from purified peripheral bloodlymphocytes (PBL) obtained from a chronic lymphocytic leukemia (CLL)patient displaying a λ light chain idiotype was extracted using theRNeasy kit (Qiagen). Approximately 2.0 μg total RNA was used as templatefor first strand cDNA synthesis using the SuperScript PreamplificationSystem (Gibco BRL) according to manufacturer's recommendation. Oligo(dT)was used for priming. One twentieth of the synthesized single strandedcDNA was amplified in a PCR reaction using an upstream primer identicalto a portion of the Vλ signal sequence (SEQ ID NO:54) and a downstreamprimer (SEQ ID NO:77) complimentary to the last several codons of theconstant region as well as a portion of the 3′ untranslated region. ThePCR products were cloned into the pCRII vector (Invitrogen) andsequenced to confirm identity. A plasmid containing the correct λconstant region sequence was chosen as a template for a second PCR. Inthis reaction a sense oligonucleotide, Cλ-5′ (SEQ ID NO:78), containingan engineered Dra III restriction site, corresponding the sequence inthe A constant region immediately downstream of Jλ and a Hind IIIcontaining antisense oligonucleotide primer, Cλ-3′ (SEQ ID NO:79)spanning the STOP codon immediately following the λ constant region wereutilized. The resulting PCR product was cloned into the pCR2.1-TOPOvector and sequenced. A fragment containing the λ constant regionsequence was released upon Hind III restriction from some of theplasmids, depending on orientation of the insert. This restrictionfragment was gel isolated and cloned into pTRABacHuLCKHCγ1 (FIG. 5A),following linearization following Hind III digestion, generating anintermediate plasmid containing both the λ and K constant regions.Restriction of this plasmid with Stu I and Dra III resulted in theremoval of the K sequences. This linearized plasmid was then ligatedwith annealed complimentary primers λ-stuff 1 (SEQ ID NO:80) and λ-stuff1′ to generate the final version of pTRABacHuLCλHCγ1 (FIG. 5B).

3. Insertion of Genes for Patient-Derived Idiotype V_(H) ND/or V_(L)Regions into an Expression Vector

Using either pTRABacHuLC_(K)HC_(γ1) or pTRABacHuLC_(λ)HC_(γ1), it waspossible to insert genes for any V_(L) region containing the uniquecloning sequences Stu I and Dra III between the alkaline phosphatasesignal sequence and the K or λ constant region, and genes for any V_(H)region containing the unique cloning sequences Spe I and Apa I betweenthe melittin secretory signal sequence and the IgG_(γ1) constant region(See FIGS. 5A and 5B). The resulting expression vector could then beutilized for transduction into Spodoptera frugiperda (Sf-9) insect cellsto produce recombinant budded baculovirus. The recombinant baculoviruswas then serially amplified in Sf-9 cells to produce a high titerrecombinant baculovirus stock. This high titer recombinant baculovirusstock was then used to infect Trichoplusia ni (High-5) cells forsubsequent chimeric IgG protein production. A list of alloligonucleotide primers used in the construction ofpTRABacHuLC_(K)HC_(γ1) or pTRABacHuLC_(λ)HC_(λ1) can be found in Table2.

After the tumor derived sequences for V_(H) and/or V_(L) regions areisolated as described above, oligonucleotide primers including theterminal 40 nucleotides of the melittin leader peptide (for Ig heavychain cloning) (SEQ ID NO:8—CAGATCACTA GTTTTTATGG TCGTGTACAT TTCTTACATCTATGCG], the terminal 28 nucleotides of the alkaline phosphatase leaderpeptide (for Ig light chain cloning) (SEQ ID NO:9—CTGAGTAGGC CTGAGGCTACAGCTCTCCCT GGGC), and the first 21 to 24 nucleotides of the respectiveV_(H) or V_(L) region proteins are prepared. Reverse oligonucleotideprimers from the heavy or light chain constant region are used (IgG: SEQID NO:10—GGAAGTAGTC CTTGACCAGG CAG; IgM: SEQ ID NO:11—GGGAAAAGGGTTGGGCCCGA TGCAC; IgK: SEQ ID NO:12—GATGAAGACA CTTGGTGCAG CCACAG; Igλ:SEQ ID NO:13: GGAACAGAGT GACACTGGGT GCAGCCTTGG GCTG). Recombinantplasmids identified previously as having the clonal V_(H) or V_(L)sequences are used as templates for a second round of PCR. Cyclingconditions were as described supra.

Plasmid templates were combined with an IgG_(γ1), IgM, Igλ, or IgKconstant region primer complementary to codon encoding amino acids141-149, 115-123, 108-119, and 109-117 respectively and the appropriateleader/V region fusion primer. For example, for one patient, the primersused were SEQ ID NO:67 for V_(H3) and SEQ ID NO:68 for V_(K3)(SEQ IDNO:67: CAGATCACTA GTTTTTATGG TCGTGTACAT TTCTTACATC TATGCGGAGA TGAAATTGGTGGAGTCTGGG; SEQ ID NO:68: CTGAGTAGGC CTGAGGCTAC AGCTCTCCCT GGGCGAAGTTGTGTTGACTC AGTCTCC). Cycling conditions were as described above.

a. Light Chain Variable Region Insertion into Expression Vector: A PCRderived V_(L) product and 2 μg of the correspondingpTRABacHuLC_(K)HC_(γ1) or pTRABacHuLC_(λ)HC_(γ1) cassette vectordigested with Stu I and Dra Ill. The 350 bp DNA fragment from thepatient derived V_(L) region and the 8.4 kb fragment for the linearpTRABacHuLC_(K)HC_(γ1) or pTRABacHuLC_(λ)HC_(γ1) vector were purifiedfrom agarose gel plugs with Qiaex II resin and eluted in 50 μl water.The DNA concentrations for both fragments were determined and then thefragments ligated using Rapid Ligation kit (Roche). The ligationproducts were used to transform competent XL1-Blue E. coli which weresubsequently plated on a LB-carbenicillin agar plate for overnightgrowing at 37° C. Miniprep colonies were prepared and the recombinantDNA plasmids were verified by restriction analysis and sequencing. Theresulting vector designated pTRABac(NHL-V_(L))LC_(K)HC_(γ1) orpTRABac(NHL-V_(L))LC_(λ)HC_(γ1).

b. Heave Chain Variable Regon Insertion into Expression Vector: A PCRderived V_(H) product and 2 μg of the pTRABac(NHL-V_(L))LC_(K) HC_(γ1)or pTRABac(NHL-V_(L))LC_(λ)HC_(γ1) cassette vector were digested withSpe I and Apa I. The 350 bp DNA fragment from the patient derived V_(H)region and the 8.8 kb fragment for the linear pTRABac(NHL-V_(L))LC_(K)HC_(γ1) or pTRABac(NHL-V_(L))LC_(λ)HC_(γ1) vector were purified fromagarose gel plugs with Qiaex II resin and eluted in 50 μl water. The DNAconcentrations for both fragments were determined and then the fragmentsligated using Rapid Ligation kit (Roche). The ligation products wereused to transform competent XL1-Blue E. coli which were subsequentlyplated on a LB-carbenicillin agar plate for overnight growing at 37° C.Miniprep colonies were prepared and the recombinant DNA plasmids wereverified by restriction analysis and sequencing. The resulting vector isdesignated pTRABac(NHL-V_(L))LC_(K)(NHL-V_(H))HC_(7i),pTRABac(NHL-V_(L))LC_(λ) (NHL-V_(H))HC_(γ1) and is assigned a referencenumber corresponding to a patient, e.g., FV8786-001.

TABLE 2Primer Sequences Used for Construction of pTRABacHuLC_(K)HC_(γ1) andpTRABacHuLC_(λ)HC_(γ1) Baculovirus Transfer Vectors. PRIMER NAMEPRIMER SEQUENCE (5′ 3′) 1. Melittin N-terminusACTAGTGCAACGTTGACTAAGAATTTCAT (MelS/N and MelN/S) GCGGCCGC(SEQ ID NO: 15) GCGGCCGCATGAAATTCTTAGTCAA-CGTT GCACTAGT (SEQ ID NO: 16)2. Human Placental GCGGATCCATGGTGGGACCCTGCATGCTG Alkaline PhosphataseCTGCTGCTGCTGCTGCTAGGCCTggaatt N- terminus (APB/E and CC APE/B)(SEQ ID NO: 17) GGAATTCCAGGCCTAGCAGCAGCAGCAGCAGCAGCATGCAGGGTCCCACCATGGATCC GC (SEQ ID NO: 18) 3. IgG_(γ) Heavy ChainTGTGACTAGTATGTATCGGCCCATCGGTC Constant: Upstream TTCCCCCT Downstream(SEQ ID NO: 19) TTTCTAGACTATTATTTACCCGGAGACAG GGAGAG (SEQ ID NO: 20)4. Kappa Light Chain CTAGGCCTATGTATCACCAAGTGTCTTCA Constant: UpstreamTCTTCCCGCCATCT  Downstream (SEQ ID NO: 21) CCCAAGCTTCTATTAACACTCTCCCCTGTTTGAAGCT (SEQ ID NO: 22)

4. Transfection of Insect Cell Lines with Variable Region-ContainingExpression Vectors and Production of Recombinant Chimeric Proteins:

a. Insect Cell Growth: Two established insect cell lines (Sf9 andHigh-5) were transfected with modified baculoviral vectors to producerecombinant chimeric V_(H)/immunoglobulin and/or V_(L)/immunoglobulinproteins. All insect cells were grown at 28° C. in ESF-921 Serum FreeInsect Media (Expression Systems LLP) containing 50 μg/L gentamycin indisposable sterile vented shaker flasks (Coming), at 140-150 rpm, withno more than 50% liquid volume. Cells were passaged every 2 to 3 days.Frozen cells were thawed (Cryo-preservation media: 10% DMSO, 40% ESF-921medium, 50% High-5 conditioned media) from a working cell bank for eachlot of product or every six weeks to assure a continuous stock ofexponentially growing cells that was not retractile to infection bybaculovirus.

b. Sf9 Cell Transfection and Recombination Assay: The modifiedbaculovirus expression vectors containing genes for V_(H) and/or V_(L)regions and genes encoding immunoglobulin heavy and/or light chainconstant regions were co-transfected into Sf9 cells using theBacVector-3000 transfection kit (Invitrogen). Ten individual plaques arepicked from agarose overlays. Virus from isolated plaques are used toinfect T-25 flasks seeded with Sf-9 cells at 50% confluency in 5 mlESF-921 media. Clonal viral isolates amplified in T-25 flasks are testedby PCR, using two primers (SEQ ID NO:36—TTTACTGTTT TCGTAACAGT TTTG) and(SEQ ID NO:37—GGTCGTTAAC AATGGGGAAG CTG) to assure clonality of theisolated plaques and that there was no wild type virus contamination. Ingeneral, 200 ng recombinant transfer vector plasmid was co-transfectedwith triple-cut Bac-Vector-3000 as described in the Bac Vector manual(Novagen) using the Eufectin lipid reagent supplied. This transfectionmixture was subjected to serially 5-fold dilutions. One hundredmicroliter aliquots were plated in 60 mm tissue culture dishescontaining 2.5×10⁶ adherent Sf9 cells. After 1 hour, cells were overlaidwith 4 ml of a 1% agarose solution in ESF-921 culture medium. Tenindividual clones were picked from the transfected cells grown inagarose overlays after staining for live cells using Neutral Red (Sigma,St. Louis, Mo.) at t=144 hours post transfection. Virus was eluted fromplaque plugs overnight in 1 ml ESF-921 media. T-25 flasks were seededwith Sf-9 cells at 50% confluency in 5 ml ESF-921 media, and infectedwith 0.5 ml of eluted clonal A virus. Ninety-six hours post infection,0.5 ml media was removed from T-25 flasks; the cells were removed bycentrifugation and the supernatant was assayed for immunoglobulinactivity by dot blotting on nitrocellulose. The absence of wild typevirus was also tested by PCR as follows.

Infectious supernatant (10 μl) containing recombinant baculovirus wasadded to 90 μl of lysis buffer containing 10 mM Tris pH 8.3, 50 mM KCl,0.1 mg/ml gelatin, 0.45% Nonidet P-40, and 0.45% Tween-20, containing 6μg Proteinase-K. The mixture was heated for 1 hour at 60° C. and theProteinase-K was denatured by incubation at 95° C. for 10 min. Twentyfive ill of the heated mixture was removed to a fresh PCR tube aftercooling, and another 25 μl of the mixture containing 10 mM Tris pH 8.3,50 mM KCl, 0.1 mg/ml gelatin, 0.45% NP-40, 0.45% Tween-20, 400 μM eachdNTP, 5 mM MgCl₂, 50 pM each PCR primher (final), and 2.5 U Taqpolymerase (Roche) was added. The viral DNA was amplified for 40 cyclesat: 92° C. for 1 min., followed by 58° C. for 1 min. and 72° C. for 1min. The recombinant baculovirus primers PH forward (SEQ ID NO:36) andPH reverse (SEQ ID NO:37) were used to amplify the polyhedron locusexpressing the light chain gene. PCR products were analyzed followingelectrophoresis through an agarose gel. Recombinant baculovirus wouldamplify a 1300 bp fragment, while wild type baculovirus would produce a˜800 bp fragment with these primer sets. Recombinant virus contaminatedwith wild type virus would amplify both fragment sizes.

C. Preparation of High Titer Viral Stocks in Sf9 Insect Cells: Two mlfrom a T-25 primary culture was transferred to a T-75 flask containingSUP cells at 50% confluency in 10 ml ESF-921 media, and cells were grownfor 120 hours at 28° C. Five ml of secondary T-75 cultures wastransferred to a 150 ml shaker flask containing 50 ml of Sf9 cells at2×10⁶ cells/ml, and cells were grown for 120 hours at 28° C. 25 ml wastransferred from the 150 ml shaker flask into 500-ml of Sf-9 cells at2×10⁶ cells/ml in a one liter shaker flask, and was grown at 28° C. Whenthe cultures reached 20%, viable cells as determined by trypan bluestaining (approximately 120 to 144 hours post infection), the viralculture was harvested by centrifugation at 3000×g, distributed into 50ml sterile tubes, and half of the tubes were stored at 4° C. with therest at −80° C. This harvested 500 ml high titer (>1×10⁸ pfu/ml) viralstock was then used to infect High-5 insect cells for immunoglobulinproduction. Viral titers (pfu/ml) were determined using a BaculovirusRapid Titer Kit (Clontech, Palo Alto, Calif.).

d. Production of Id in High-5 Insect Cells: High-5 insect celis(BTI-TN-5B1-4) secreted higher levels (2-20×) of recombinantimmunoglobulin compared to Sip cells, and were chosen for chimericprotein production. Early log phase High-5 cells (1.0-2.0×10⁶ cells/ml)were seeded in 1 liter disposable culture flasks with vented closures at5×10⁵ cells/ml in ESF921 Media (Expression Systems LLP). The flasks wereshaken at 140-150 rpm at 28° C., and the volume of media in the flaskswas adjusted over time to no greater than 500 ml. When the celldensities reached 1.5-2.5 cell/ml in 500 ml media, the flasks wereinfected with high titer recombinant baculovirus stock at a multiplicityof infection (MOI) approximating 0.5:1 (pfi:cells). The flasks were thenshaken at 140-150 rpm at 28° C.; the culture was harvested 96 hourspost-infection.

5. Purification of the Chimeric Protein Comprising a V_(H),Immunoglobulin and a V_(L)-Immunoglobulin:

Cells and debris were removed by centrifugation for 60 min. atapproximately 5,000×g, followed by filtration through a 0.2μ PES sterilefilter unit. Chimeric proteins were purified from cleared tissue culturemedia by affinity chromatography with a Protein-A High-Trap cartridge(Amersham Pharmacia, Piscataway, N.J.), followed by ion-exchangechromatography utilizing FPLC technology (Amersham Pharmacia). Thepurified chimeric proteins were size separated and buffer exchanged intoPBS by FPLC chromatography. All reagents used for protein purificationwere of USP biotechnology grade (GenAr, Mallinckrot Baker, Parris, Ky.)and endotoxin tested by the manufacturer. Sterile USP grade water wasused to make all buffers and other solutions. Buffers and othersolutions were prepared in a biological safety cabinet, and filtersterilized through 0.2 μm PES filter units.

a. Protein A Sepharose Affinity Purification of the Chimeric Proteins:

Tissue culture medium was removed from growing culture flasks and spunfor 60 min. at 5,000×g to sediment cells and debris. The supernatant wassterilized by filtration using a 0.2μ PES filter unit. Tris buffer (1M,pH 7.4) was added to the filtered medium containing V_(H) and/orV_(L)-immunoglobulin chimeric proteins to a final concentration of 20mM. The buffered tissue culture supernatant was loaded onto a 5 mlHighTrap recombinant Protein A Sepharose affinity cartridge at a flowrate of 1 to 5 ml/min with a P1 peristaltic pump (Amersham Pharmacia)collecting the flow-through in a clean flask. The column was washed with25 ml PBS (pH 7.4) at 5 ml/min. The direction of the flow was reversedand the column was washed with an additional 25 ml PBS. The column waseluted in reverse at 1 ml/min with 0.05 M citric acid (pH 3.5)collecting 1 ml fractions. Other protein columns including but notlimited to protein G, protein L, or any proteins that are-able to bindto an immunoglobulin binding domain could be used in the same manner.

b. Ion Exchange Chromatography: A 5 ml High Trap SP Sepharose cationexchange cartridge was equilibrated with 50 ml of 25 mM citric acid (pH3.5) and 20 mM NaCl. The Protein A eluted V_(H) and/or V_(L)-IgGchimeric proteins were loaded directly onto the equilibrated High TrapSP Sepharose column using a peristaltic pump at a flow rate of 1 ml/min.The column was washed with 25 ml 50 mM citric acid (pH 3.5) and 20 mMNaCl (Buffer A) at 2 ml/min. The column was eluted with a lineargradient (0% Buffer B to 100% Buffer B) to collect 1 ml fractions at 1ml/min. (Buffer B=100 mM Na carbonate (pH 10.0) and 1M NaCl). The ionexchange eluted fractions containing V_(H) and/or V_(L)-IgG chimericproteins were analyzed spectrophotometrically by their OD₂₈₀. The peakfractions were pooled.

C. Size Exclusion Chromatography: The pooled Ig fraction from SPion-exchange was then loaded onto a Hi Prep Sephacryl 26/60 S200 HiResolution column (Pharmacia) that had been equilibrated in 5 columnvolumes of PBS (pH7.2) following a pre-wash in 100 ml sterile water. Thechimeric Ig proteins were eluted in PBS at a flow rate of 0.5 ml/min andcollected in 1 ml fractions. The major Ig peak was apooled a sterilefiltered through a 0.2μ filter.

6. Idiotypic Protein and Keyhole Limpet Hemocyanin (KLH) Conjugation.

Once purified, the idiotypic protein was conjugated to GMP grade KLH(VACMUNE, Biosyn Corporation) via glutaraldehyde crosslinking. At least5 mg of purified, sterile idiotypic protein as described, supra, wascombined with an equal weight of KLH in a sterile 15 ml conical tube andthe final volume was adjusted to 9 ml in PBS. One ml of 1%glutaraldehyde (25% Grade 1 aqueous solution, Sigma) was added dropwiseto a final concentration of 0.1%. The tube was then slowly rocked for 4hours at room temperature. The conjugate was dialyzed in sterileDispoDialyzers (Spectrum Labs) against 2 liters sterile PBS, with threebuffer changes over at least 24 hours in a biological safety hood. Thefinal IgG/KLH conjugate in PBS is aseptically removed from the dialysischambers and transferred into a sterile tube, mixed, then aliquoted invials. Each vial of final product was labeled with the lot number,patient identifier, vial number and date vialed. Ten percent of thefinal vialed lot was tested for sterility and a vial was tested for thepresence of endotoxin. One vial was retained for archival purposes.

7. Product Tests

a. DNA Sequence of Baculovirus Containing Production Lot

Supernantant: A 1 ml aliquot of sample of infected insect cellproduction culture supernatant was harvested and cleared of cellulardebris by spinning at 3000 rpm for 5 min in a desktop centrifuge. Atleast 0.1 ml of this cleared supernatant containing baculovirusparticles was combined at a volume ratio of 1 to 9 with lysis buffer (10mM Tris pH 8.3, 50 mM KCl, 0.1 mg/ml gelatin, 0.45% Nonidet P-40, and0.45% Tween-20), subjected to proteolysis with proteinase K (finalconcentration 60 μg/ml) for 1 h at 60° C., followed by denaturation for15 min at 95° C. Twenty-five μl of this lysate was then combined with anadditional 25 μl of the above lysis buffer containing 400 μM each dNTP,5 MM MgCl₂, 25 pmol forward and reverse oligonucleotide primers (seeTable 3; SEQ ID NO:34 and SEQ ID NO:31 for V_(H) Identification and SEQID NO:35 and SEQ ID NO:36 for V_(L) identification, respectively), and2.5 U Taq polymerase (Roche). Cycling conditions for the PCR of V_(L)are: initial denaturation for 2 min at 92° C., followed by 40 cycles of1 min each at 92° C., 58° C., and 72° C., with a final extension of 7min at 72° C. For the PCR of V_(H), cycling conditions are the sameexcept that the annealing temperature is 64° C. PCR products wereassessed for expected size and quantity by agarose gel electrophoresis.Subsequently, two or more nested primers were used to directly sequencethe PCR products. (See Table 3; SEQ ID NO:30 and SEQ ID NO:34 for V_(H)identification, SEQ ID NO:28 and SEQ ID NO:35 for VK identification, andSEQ ID NO:88 and SEQ ID NO:35 for Vλ identification, respectively.) Thecomplete V_(H) and V_(L) nucleotide sequences was determined using theOpenGene Automated DNA Sequencing System (Visible Genetics) andsequencing analysis software, as described above and compared with theV-gene sequences of the pTRABac(NHL-FV-8786-XXX) vector corresponding tothat patient's idiotype.

b. Superose 6 Gel Filtration Chromatography: Gel filtrationchromatography of the purified Id was performed to assess proteinpurity. Gel filtration chromatography was performed using a Superose 6HR 10/30 FPLC column (Amersham Pharmacia) with PBS as the liquid phase.Peak integration was performed on the largest 20 peaks by the FPLCsoftware using the following criteria to reject a peak from beingincluded in area evaluation: height less than 0.01 Au; width less than0.05 ml; area less than 0.01 Au/ml. Fractions of each column run werecollected and assayed for human immunoglobulin specific activity bycapture ELISA, and compared to the OD₂₈₀ chromatogram.

c. Immunoglobulin Assay; Anti Human IgG ELISA: Microtiter plate wellswere coated with 100 μl of a 3 μg/ml dilution of Goat anti-Human IgGheavy chain specific antibody (Roche) in carbonate buffer overnight at4° C., and washed 2 times with 100 μl TBS (50 mM Tris, 150 mM NaCl, pH7.5). Wells were blocked with of 200 μl TBSB (TBS+1% BSA) for 1 hour at22° C.

Each chromatogram fraction corresponding to human peak in TBSB wastested. One hundred μl of diluted sample was added in 2-fold serialdilutions to wells in replicates, and incubated 1 hour at 22° C. Theassay was repeated with purified Human IgG1/K or IgG1λ standards (Sigma,St. Louis, Mo.). The wells were washed 4 times with 200 TBST (TBS+0.1%Tween 20). The detection antibody was diluted (Goat-anti-Human K orλ-HRP (Fischer, Pittsburgh, Pa.) 1:2000 in TBSB, 100 μl was added towells, and incubated for 1 hour at 22° C. The wells were washed 6 timeswith 200 μl TBST. One hundred μl of substrate (TMB 1 component, KPLInc., Gaithersburg, Md.) was added to wells, developed 30 min. andassayed at OD₆₂₀.

d. Idiotypic Protein Release Criteria: (1) The DNA sequence ofidiotype-variable genes in baculovirus from production supernantant mustbe identical to the DNA sequence in the production vector. (2) Theidiotypic protein concentration was greater than 0.5 mg/ml based onOD₂₈₀. (3) The major peak area was greater than 90% of area in evaluatedpeaks on Superose 6 analytical chromatography. (4) The majorchromatographic peak corresponds to the human IgGK (or λ) ELISA activitypeak.

The final vaccine product, Id-KLH, was tested for endotoxin levels by akinetic turbidity microplate assay or a Limulus Amoebocyte Lysate (LAL)assay and had a level below 350 endotoxin units (EU) per ml. Ten percentof the lot was tested for sterility on a 14-day test and tests negativeor was discarded.

Table 3 shows a summary of primer sequences used for establishing finalproduct identity.

TABLE 3 Primer Sequences Used for Establishing Final Product IdentityPRIMER NAME PRIMER SEQUENCE (5′ 3′) 1. Human Placental AlkalineAAATGATAACCATCTCGC Phosphatase Internal (SEQ ID NO: 25)2. Human Placental Alkaline TTTACTGTTTTCGTAACAGTTTTGPhosphatase External (SEQ ID NO: 26) 3. Kappa Light Chain ConstantTTGGAGGGCGTTATCCACCTTC Antisense (SEQ ID NO: 27)4. Kappa Light Chain Constant CTGTAAATCAACAACGCACAG Downstream Internal(SEQ ID NO: 28) 5. Kappa Light Chain Constant CAACAACGCACAGAATCTAGDownstream External (SEQ ID NO: 29) 6. Melittin InternalGGGACCTTTAATTCAACCCAACAC (SEQ ID NO: 30) 7. Melittin ExternalAAACGCGTTGGAGTCTTGTGTGC (SEQ ID NO: 31) 8. IgG_(γ1) Heavy Chain ConstantGGAAGTAGTCCTTGACCAGGCAG Downstream Internal (SEQ ID NO: 32)9. IgG_(γ1) Heavy Chain Constant CTGAGTTCCACGACACCGTCACDownstream Middle (SEQ ID NO: 33) 10. IgG_(γ1) Heavy Chain ConstantTAGAGTCCTGAGGACTGTAGGAC Downstream External (SEQ ID NO: 34)11. Kappa & Lambda Downstream: 5′-GGTCGTTAACAATGGGGAAGCTG-3′(SEQ ID NO: 35) 12. PH forward 5′-TTTACTGTTTTCGTAACAGTTTTG-3′(SEQ ID NO: 36) 13. PH reverse 5′-GGTCGTTAACAATGGGGAAGCTG-3′(SEQ ID NO: 37) 14. Lambda Constant Internal5′-GAAGTCACTTATGAGACACACCAG-3′ (SEQ ID NO: 38)

8. Use of Chimeric Protein of the Invention for Treatment ofNon-Hodgkin's B-Cell Lymphoma.

V_(H) and V_(L) regions were obtained from a patient with Non-Hodgkin'sB-Cell Lymphoma. Using the 5′ RACE method described supra, genesencoding these regions were cloned and inserted into the expressionvector and expressed by the methods of the instant invention. Table 5contains the DNA sequences of the Vh and Vl regions used for theexpression vector. The Apa I and Dra III sites used for cloning areindicated by underlining.

TABLE 5 Variable region sequences obtained from  a patient. VH A/07SEQ ID NO: 86 GACATGTTGTTGGTGGAATCGGGGGG-AGGCCTGGTCCAGCCGGGGGAGTCCCTGAGACTCTCCTGTGTGGCCTCTAGATTCACCTTTAGAAC-GTTTTGGATGACCTGGGTCCGCCAACTTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATATAAATCAAGATGGCAGTCAGACGTATCATGCGGACTCTGTAAAGGGCCGATTTACCATCTCCAGAGACAACGGCAGGAACTCCCTATTTTTACAAATGACAAGTCTGAGAGTCGCGGACACGGCTATATATTACTGTGCGACTAATGAAACGTCCAGTGGCCTGGACTGCTGGGGCCAAGGAACCCTGGTCACTGTCTCCTCAGC TTCCACCAAGGGCCC VK A/L6SEQ ID NO: 87 GAAATCGTGTTGACACAGTCTCCAGCCACCCTGTCTTCGTCTCCAGGAGACAGAGTCGCCCTCTCCTGCAGGGCCAGTCAGAGTGTAAGAAGTTACTTAAGTTGGTATCAACAGAAGGCTGGCCAGGCTCCCAGGCTCCTCATCCATAATGCATCCAGTAGGGCCACTGGCATCCCGCCCAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGTCGCCTAGAGACTGAAGATGCTGCAGTTTATTACTGTCAGCAACTTTATTTCTGGCCTCCGATATTATTTTTCGGCCCTGGGACCAAAGTGAA TATCACACGAACTGTGGCTGCACCAAGTG

The isolated recombinant chimeric immunoglobulin protein produced forthis patient from the genetic information detailed above was conjugatedto KLH and administered with GM-CSF five times over a six-month periodas described supra. A CT scan was performed on the neck and pelvis areasof the patient prior to administration of the therapy and 9 monthslater. A comparison of the sum of the diameters of 6 tumor massesrevealed a 60% reduction nine months following therapy initiation. (Notethat these figures are not adjusted to accommodate the size of the lymphnode prior to diagnosis of the disease (See, Cheson et al., J. Clin.Oncol., 17(4):1244, 1999.)

TABLE 6 Reduction in size of lymph nodes following treatment. PRIOR TOTXT. (Product of 9 MONTHS POST TXT. diameters; cm²) (Product ofdiameters; cm²) LYMPH NODE 1 6.16 2.8 LYMPH NODE 2 5.0 1.6 LYMPH NODE 33.3 1.17 LYMPH NODE 4 3.78 1.44 LYMPH NODE 5 1.92 1.0 LYMPH NODE 6 1.080.80 SUM OF DIAMETERS 21.24 8.81

What is claimed is:
 1. A composition comprising two different proteinsproduced by a process comprising the steps of: (a) isolating a geneencoding a full length sequence of a human V_(H) region isolated from aB cell clone of a human patient having a B cell mediated pathology; (b)linking said gene encoding said entire V_(H) region and a gene encodingat least a portion of an immunoglobulin constant region in an expressionvector to form a gene encoding the first of said different proteins; (c)isolating a gene encoding a full length sequence of a human V_(L) regionisolated from a B cell clone of said patient having said B cell mediatedpathology; (d) linking said gene encoding said entire V_(L) region and agene encoding at least a portion of an immunoglobulin constant regioninto said expression vector to form a gene encoding the second of saiddifferent proteins; (e) introducing said expression vector into insectcells, wherein said expression vector contains two separate expressioncassettes each having a baculovirus promoter and a heterologoussecretory signal sequence; and (f) allowing the expression of said twodifferent proteins having the same V_(H) and V_(L) amino acid sequencesin said patient, wherein said composition further comprises a cytokineor chemokine, and wherein said composition elicits an active immuneresponse.
 2. The composition of claim 1 wherein said carrier protein isa keyhole-limpet hemocyanin (KLH).
 3. The composition of claim 1,wherein said composition is administered to said patient with agranulocyte-macrophage-colony stimulating factor (GM-CSF) cytokine. 4.The composition of claim 1, wherein said first protein and/or saidsecond protein is further conjugated to a carrier protein
 5. Thecomposition of claim 1, wherein said gene encoding said first of saidproteins comprising said entire V_(H) region and a first immunoglobulinconstant region is controlled by a p10 promoter in an expression vector,and said gene encoding said second of said proteins comprising saidentire V_(L) region and a second first immunoglobulin constant region iscontrolled by a polyhedrin promoter in an expression vector.
 6. Thecomposition of claim 1, wherein one of said genes encoding saidimmunoglobulin constant regions is a gene encoding at least a portion ofa human κ or λ constant region.
 7. The composition of claim 1, whereinsaid gene encoding one of said immunoglobulin constant regions is a geneencoding at least a portion of an immunoglobulin constant regionselected from the group consisting of a human IgGγ1 constant region, ahuman IgGγ2 constant region, a human IgGγ3 constant region, a humanIgGγ4 constant region, a human IgA1 constant region, a human IgA2constant region, a human IgM constant region, a human IgD constantregion, a human IgE constant region, a human κ, chain constant region,and a human λ chain constant region.
 8. The composition of claim 1,wherein said proteins are used to treat refractory low grade lymphoma orfollicular B cell lymphoma.
 9. The composition of claim 1, wherein saidproteins are produced in insect cells selected from the group consistingof the Trichoplusia ni cell line and the Spodoptera frugiperda cellline, and wherein said expression vector is a baculovirus expressionvector that comprises a honey bee melittin secretory signal sequence anda human placental alkaline phosphatase secretory signal sequence. 10.The composition of claim 1, wherein said expression vector is abaculovirus expression vector that comprises a baculovirus p10 promoterand polyhedrin promoter, wherein said p10 promoter controls a honey beemelittin secretory signal sequence, and wherein said polyhedrin promotercontrols a human placental alkaline phosphatase secretory signalsequence.
 11. A composition comprising two separate and distinctproteins produced by a process comprising the steps of: (a) isolating agene encoding a full length sequence of a human V_(H) region isolatedfrom a B cell clone of a human patient having a B cell mediatedpathology; (b) linking said gene encoding said entire V_(H) region and agene encoding at least a portion of an immunoglobulin constant region inan expression vector to form a gene encoding the first of said separateand distinct proteins; (c) isolating a gene encoding a full lengthsequence of a human V_(L) region isolated from a B cell clone of saidpatient having said B cell mediated pathology; (d) linking said geneencoding said entire V_(L) region and a gene encoding at least a portionof an immunoglobulin constant region into an expression vector to form agene encoding the second of said separate and distinct proteins; (e)introducing said expression vectors into insect cells, wherein saidexpression vectors contain two separate expression cassettes each havinga baculovirus promoter and a heterologous secretory signal sequence,wherein (i) said promoters are selected from the group consisting of p10promoter and polyhedrin promoter, and (ii) said secretory signalsequences are selected from the group consisting of human placentalalkaline phosphatase secretory signal sequence, honey bee melittinsecretory signal sequence, and the endogenous secretory signal sequenceassociated with the immunoglobulin genes isolated from said patient; and(f) allowing the expression of said two separate and distinct proteinshaving the same V_(H) and V_(L) amino acid sequences in said patient.12. The composition of claim 11, wherein said expression vectors arebaculovirus expression vectors.
 13. The composition of claim 11, whereinsaid expression vector is a baculovirus expression vector that comprisesa baculovirus p10 promoter and polyhedrin promoter, wherein said p10promoter controls a honey bee melittin secretory signal sequence, andwherein said polyhedrin promoter controls a human placental alkalinephosphatase secretory signal sequence.
 14. The composition of claim 11,wherein said first protein and/or said second protein is furtherconjugated to a carrier protein and is administered to said patienttogether with a cytokine or chemokine, and wherein said compositionelicits an active immune response.
 15. The composition of claim 11,wherein said proteins are conjugated to a keyhole-limpet hemocyanin(KLH).
 16. The composition of claim 11, wherein said composition isadministered to said patient.
 17. The composition of claim 16, whereinsaid composition is administered to said patient with a monocytechemotactic protein 3 (MCR 3) chemokine.
 18. The composition of claim17, wherein said composition is administered to said patient with agranulocyte-macrophage-colony stimulating factor (GM-CSF) cytokine. 19.The composition of claim 11, wherein said gene encoding said first ofthe proteins comprising a full length V_(H) region and a firstimmunoglobulin constant region is controlled by a p10 promoter, and saidgene encoding said second of the proteins comprising a full length V_(L)region and a second first immunoglobulin constant region is controlledby a polyhedrin promoter.
 20. The composition of claim 11, wherein oneof said genes encoding at least a portion of said immunoglobulinconstant regions is a gene encoding at least a portion of a human κ or λconstant region, and wherein said gene encoding at least a portion ofsaid immunoglobulin constant regions is a gene encoding at least aportion of an immunoglobulin constant region selected from the groupconsisting of a human IgGγ1 constant region, a human IgGγ2 constantregion, a human IgGγ3 constant region, a human IgGγ4 constant region, ahuman IgA1 constant region, a human IgA2 constant region, a human IgMconstant region, a human IgD constant region, a human IgE constantregion, a human k chain constant region, and a human 1 chain constantregion.